RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)

ABSTRACT

The present invention concerns methods and reagents useful in modulating gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to synthetic chemically modified small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against target nucleic acid sequences. The small nucleic acid molecules are useful in the treatment of any disease or condition that responds to modulation of gene expression or activity in a cell, tissue, or organism.

This application is a continuation of U.S. patent application Ser. No.16/136,191, filed Sep. 19, 2018, which is a continuation of U.S. patentapplication Ser. No. 15/977,746, filed May 11, 2018, which is acontinuation of U.S. patent application Ser. No. 15/637,579, filed Jun.29, 2017 (now issued as U.S. Pat. No. 10,000,754), which is acontinuation of U.S. patent application Ser. No. 15/084,865, filed Mar.30, 2016 (now issued as U.S. Pat. No. 9,732,344), which is acontinuation of U.S. patent application Ser. No. 14/861,805, filed Sep.22, 2015 (now issued as U.S. Pat. No. 9,657,294), which is acontinuation of U.S. patent application Ser. No. 14/514,112, filed Oct.14, 2014 (now issued as U.S. Pat. No. 9,181,551), which is acontinuation of U.S. patent application Ser. No. 14/083,525, filed Nov.19, 2013 (now abandoned), which is a continuation of U.S. patentapplication Ser. No. 13/480,655 filed May 25, 2012 (now issued as U.S.Pat. No. 8,618,277), which is a continuation of U.S. patent applicationSer. No. 10/720,448 filed Nov. 24, 2003 (now issued as U.S. Pat. No.8,273,866), which is a continuation-in-part of U.S. patent applicationSer. No. 10/693,059, filed Oct. 23, 2003 (now abandoned), which is acontinuation-in-part of U.S. patent application Ser. No. 10/444,853,filed May 23, 2003 (now issued as U.S. Pat. No. 8,202,979) and acontinuation-in-part of Ser. No. 10/652,791, filed Aug. 29, 2003 (nowabandoned). The Ser. No. 10/693,059 application is also acontinuation-in-part of International Patent Application No.PCT/US2003/005346, filed Feb. 20, 2003, and a continuation-in-part ofInternational Patent Application No. PCT/US03/05028, filed Feb. 20,2003, both of which claim the benefit of U.S. Provisional ApplicationNo. 60/358,580 filed Feb. 20, 2002, U.S. Provisional Application No.60/363,124 filed Mar. 11, 2002, U.S. Provisional Application No.60/386,782 filed Jun. 6, 2002, U.S. Provisional Application No.60/406,784 filed Aug. 29, 2002, U.S. Provisional Application No.60/408,378 filed Sep. 5, 2002, U.S. Provisional Application No.60/409,293 filed Sep. 9, 2002, and U.S. Provisional Application No.60/440,129 filed Jan. 15, 2003. The Ser. No. 10/693,059 application isalso a continuation-in-part of U.S. patent application Ser. No.10/427,160, filed Apr. 30, 2003 (now issued as U.S. Pat. No. 7,833,992).The instant application claims the benefit of all the listedapplications, which are hereby incorporated by reference herein in theirentireties, including the drawings.

FIELD OF THE INVENTION

The present invention concerns methods and reagents useful in modulatinggene expression in a variety of applications, including use intherapeutic, diagnostic, target validation, and genomic discoveryapplications. Specifically, the invention relates to synthetic smallnucleic acid molecules, such as short interfering nucleic acid (siNA),short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA(miRNA), and short hairpin RNA (shRNA) molecules capable of mediatingRNA interference (RNAi).

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created in Jun. 4, 2014, isnamed A2038-720624_SL.txt and is 203,137 bytes in size.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention. Applicant demonstratesherein that chemically modified short interfering nucleic acids possessthe same capacity to mediate RNAi as do siRNA molecules and are expectedto possess improved stability and activity in vivo; therefore, thisdiscussion is not meant to be limiting only to siRNA and can be appliedto siNA as a whole.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951). The corresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response though amechanism that has yet to be fully characterized. This mechanism appearsto be different from the interferon response that results fromdsRNA-mediated activation of protein kinase PKR and 2′,5′-oligoadenylatesynthetase resulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Hamilton et al., supra; Zamore et al., 2000,Cell, 101, 25-33; Berstein et al., 2001, Nature, 409, 363). Shortinterfering RNAs derived from dicer activity are typically about 21 toabout 23 nucleotides in length and comprise about 19 base pair duplexes(Hamilton et al., supra; Elbashir et al., 2001, Genes Dev., 15, 188).Dicer has also been implicated in the excision of 21- and 22-nucleotidesmall temporal RNAs (stRNAs) from precursor RNA of conserved structurethat are implicated in translational control (Hutvagner et al., 2001,Science, 293, 834). The RNAi response also features an endonucleasecomplex, commonly referred to as an RNA-induced silencing complex(RISC), which mediates cleavage of single-stranded RNA having sequencecomplementary to the antisense strand of the siRNA duplex. Cleavage ofthe target RNA takes place in the middle of the region complementary tothe antisense strand of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494, describe RNAi induced byintroduction of duplexes of synthetic 21-nucleotide RNAs in culturedmammalian cells including human embryonic kidney and HeLa cells. Recentwork in Drosophila embryonic lysates (Elbashir et al., 2001, EMBO J.,20, 6877) has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21-nucleotidesiRNA duplexes are most active when containing 3′-terminal dinucleotideoverhangs. Furthermore, complete substitution of one or both siRNAstrands with 2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAiactivity, whereas substitution of the 3′-terminal siRNA overhangnucleotides with 2′-deoxy nucleotides (2′-H) was shown to be tolerated.Single mismatch sequences in the center of the siRNA duplex were alsoshown to abolish RNAi activity. In addition, these studies also indicatethat the position of the cleavage site in the target RNA is defined bythe 5′-end of the siRNA guide sequence rather than the 3′-end of theguide sequence (Elbashir et al., 2001, EMBO J., 20, 6877). Other studieshave indicated that a 5′-phosphate on the target-complementary strand ofa siRNA duplex is required for siRNA activity and that ATP is utilizedto maintain the 5′-phosphate moiety on the siRNA (Nykanen et al., 2001,Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877). In addition,Elbashir et al., supra, also report that substitution of siRNA with2′-O-methyl nucleotides completely abolishes RNAi activity. Li et al.,International PCT Publication No. WO 00/44914, and Beach et al.,International PCT Publication No. WO 01/68836 preliminarily suggest thatsiRNA may include modifications to either the phosphate-sugar backboneor the nucleoside to include at least one of a nitrogen or sulfurheteroatom, however, neither application postulates to what extent suchmodifications would be tolerated in siRNA molecules, nor provides anyfurther guidance or examples of such modified siRNA. Kreutzer et al.,Canadian Patent Application No. 2,359,180, also describe certainchemical modifications for use in dsRNA constructs in order tocounteract activation of double-stranded RNA-dependent protein kinasePKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotidescontaining a 2′-O or 4′-C methylene bridge. However, Kreutzer et al.similarly fails to provide examples or guidance as to what extent thesemodifications would be tolerated in siRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain dsRNA molecules into cells for use in inhibiting geneexpression. Plaetinck et al., International PCT Publication No. WO00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific dsRNA molecules. Mello et al., International PCT PublicationNo. WO 01/29058, describe the identification of specific genes involvedin dsRNA-mediated RNAi. Deschamps Depaillette et al., International PCTPublication No. WO 99/07409, describe specific compositions consistingof particular dsRNA molecules combined with certain anti-viral agents.Waterhouse et al., International PCT Publication No. 99/53050, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA constructs foruse in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically-modified siRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al., International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain dsRNAs. Echeverri et al., International PCTPublication No. WO 02/38805, describe certain C. elegans genesidentified via RNAi. Kreutzer et al., International PCT PublicationsNos. WO 02/055692, WO 02/055693, and EP 1144623 B1 describes certainmethods for inhibiting gene expression using RNAi. Graham et al.,International PCT Publications Nos. WO 99/49029 and WO 01/70949, and AU4037501 describe certain vector expressed siRNA molecules. Fire et al.,U.S. Pat. No. 6,506,559, describe certain methods for inhibiting geneexpression in vitro using certain long dsRNA (greater than 25nucleotide) constructs that mediate RNAi.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating RNA function and/or gene expression in a cell.Specifically, the instant invention features synthetic small nucleicacid molecules, such as short interfering nucleic acid (siNA), shortinterfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA),and short hairpin RNA (shRNA) molecules capable of modulating geneexpression in cells by RNA inference (RNAi). The siNA molecules of theinvention can be chemically modified. The use of chemically modifiedsiNA can improve various properties of native siRNA molecules throughincreased resistance to nuclease degradation in vivo and/or improvedcellular uptake. The chemically modified siNA molecules of the instantinvention provide useful reagents and methods for a variety oftherapeutic, diagnostic, agricultural, target validation, genomicdiscovery, genetic engineering and pharmacogenomic applications.

In a non-limiting example, the introduction of chemically modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example when compared toan all RNA nucleic acid molecule, the overall activity of the modifiednucleic acid molecule can be greater than the native molecule due toimproved stability and/or delivery of the molecule. Unlike nativeunmodified siRNA, chemically modified siNA can also minimize thepossibility of activating interferon activity in humans.

In one embodiment, the nucleic acid molecules of the invention that actas mediators of the RNA interference gene silencing response arechemically modified double stranded nucleic acid molecules. As in theirnative double stranded RNA counterparts, these siNA molecules typicallyconsist of duplexes containing about 19 base pairs betweenoligonucleotides comprising about 19 to about 25 nucleotides. The mostactive siRNA molecules are thought to have such duplexes withoverhanging ends of 1-3 nucleotides, for example 21 nucleotide duplexeswith 19 base pairs and 2 nucleotide 3′-overhangs. These overhangingsegments are readily hydrolyzed by endonucleases in vivo. Studies haveshown that replacing the 3′-overhanging segments of a 21-mer siRNAduplex having 2 nucleotide 3′ overhangs with deoxyribonucleotides doesnot have an adverse effect on RNAi activity. Replacing up to 4nucleotides on each end of the siRNA with deoxyribonucleotides has beenreported to be well tolerated whereas complete substitution withdeoxyribonucleotides results in no RNAi activity (Elbashir et al., 2001,EMBO J., 20, 6877). In addition, Elbashir et al, supra, also report thatsubstitution of siRNA with 2′-O-methyl nucleotides completely abolishesRNAi activity. Li et al., International PCT Publication No. WO 00/44914,and Beach et al., International PCT Publication No. WO 01/68836 bothsuggest that siRNA may include modifications to either thephosphate-sugar back bone or the nucleoside to include at least one of anitrogen or sulfur heteroatom, however neither application teaches towhat extent these modifications are tolerated in siRNA molecules norprovide any examples of such modified siRNA. Kreutzer and Limmer,Canadian Patent Application No. 2,359,180, also describe certainchemical modifications for use in dsRNA constructs in order tocounteract activation of double stranded-RNA-dependent protein kinasePKR, specifically 2′-amino or 2′-O-methyl nucleotides, and nucleotidescontaining a 2′-O or 4′-C methylene bridge. However, Kreutzer and Limmersimilarly fail to show to what extent these modifications are toleratedin siRNA molecules nor provide any examples of such modified siRNA.

In one embodiment, the invention features chemically modified siNAconstructs having specificity for target nucleic acid molecules in acell. Non-limiting examples of such chemical modifications includewithout limitation phosphorothioate internucleotide linkages,2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides,2′-deoxy ribonucleotides, “universal base” nucleotides, 5-C-methylnucleotides, and inverted deoxyabasic residue incorporation. Thesechemical modifications, when used in various siNA constructs, are shownto preserve RNAi activity in cells while at the same time, dramaticallyincreasing the serum stability of these compounds. Furthermore, contraryto the data published by Parrish et al., supra, applicant demonstratesthat multiple (greater than one) phosphorothioate substitutions arewell-tolerated and confer substantial increases in serum stability formodified siNA constructs.

In one embodiment, the chemically-modified siNA molecules of theinvention comprise a duplex having two strands, one or both of which canbe chemically-modified, wherein each strand is about 19 to about 29(e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. In oneembodiment, the chemically-modified siNA molecules of the inventioncomprise a duplex having two strands, one or both of which can bechemically-modified, wherein each strand is about 19 to about 23 (e.g.,about 19, 20, 21, 22, or 23) nucleotides. In one embodiment, a siNAmolecule of the invention comprises modified nucleotides whilemaintaining the ability to mediate RNAi. The modified nucleotides can beused to improve in vitro or in vivo characteristics such as stability,activity, and/or bioavailability. For example, a siNA molecule of theinvention can comprise modified nucleotides as a percentage of the totalnumber of nucleotides present in the siNA molecule. As such, a siNAmolecule of the invention can generally comprise modified nucleotidesfrom about 5 to about 100% of the nucleotide positions (e.g., 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 100% of the nucleotide positions). The actualpercentage of modified nucleotides present in a given siNA moleculedepends on the total number of nucleotides present in the siNA. If thesiNA molecule is single stranded, the percent modification can be basedupon the total number of nucleotides present in the single stranded siNAmolecules. Likewise, if the siNA molecule is double stranded, thepercent modification can be based upon the total number of nucleotidespresent in the sense strand, antisense strand, or both the sense andantisense strands. In addition, the actual percentage of modifiednucleotides present in a given siNA molecule can also depend on thetotal number of purine and pyrimidine nucleotides present in the siNA,for example, wherein all pyrimidine nucleotides and/or all purinenucleotides present in the siNA molecule are modified.

The antisense region of a siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. The antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. The 3′-terminal nucleotide overhangs of a siNAmolecule of the invention can comprise ribonucleotides ordeoxyribonucleotides that are chemically-modified at a nucleic acidsugar, base, or backbone. The 3′-terminal nucleotide overhangs cancomprise one or more universal base ribonucleotides. The 3′-terminalnucleotide overhangs can comprise one or more acyclic nucleotides.

In one embodiment, a siNA molecule of the invention comprises bluntends, i.e., the ends do not include any overhanging nucleotides. Forexample, a siNA molecule of the invention comprising modificationsdescribed herein (e.g., comprising nucleotides having Formulae I-VII orsiNA constructs comprising Stab1-Stab18 or any combination thereof)and/or any length described herein can comprise blunt ends or ends withno overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In a non-limiting example, a blunt ended siNA molecule hasa number of base pairs equal to the number of nucleotides present ineach strand of the siNA molecule. In another example, a siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, a siNA molecule comprises one bluntend, for example wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, a siNA molecule comprises two blunt ends, for examplewherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 18 to about 30nucleotides (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30 nucleotides). Other nucleotides present in a blunt ended siNAmolecule can comprise mismatches, bulges, loops, or wobble base pairs,for example, to modulate the activity of the siNA molecule to mediateRNA interference.

By “blunt ends” is meant symmetric termini or termini of a doublestranded siNA molecule having no overhanging nucleotides. The twostrands of a double stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complimentary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to down-regulateexpression of a target gene, wherein the siNA molecule comprises one ormore chemical modifications and each strand of the double-stranded siNAis about 19 to about 23 nucleotides (e.g., about 19, 20, 21, 22, or 23nucleotides) long.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene, wherein the siNA molecule comprises no ribonucleotidesand each strand of the double-stranded siNA comprises about 19 to about23 nucleotides.

In one embodiment, one of the strands of a double-stranded siNA moleculeof the invention comprises a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof of a target gene, andwherein the second strand of a double-stranded siNA molecule comprises anucleotide sequence substantially similar to the nucleotide sequence ora portion thereof of the target gene.

In one embodiment, a siNA molecule of the invention comprises about 19to about 23 nucleotides, and each strand comprises at least about 19nucleotides that are complementary to the nucleotides of the otherstrand.

In one embodiment, a siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof of a target gene, and thesiNA further comprises a sense region, wherein the sense regioncomprises a nucleotide sequence substantially similar to the nucleotidesequence or a portion thereof of the target gene. The antisense regionand the sense region each comprise about 19 to about 23 nucleotides, andthe antisense region comprises at least about 19 nucleotides that arecomplementary to nucleotides of the sense region.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence or aportion thereof of RNA encoded by a target gene and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion.

In one embodiment, a siNA molecule of the invention is assembled fromtwo separate oligonucleotide fragments wherein one fragment comprisesthe sense region and the second fragment comprises the antisense regionof the siNA molecule. In another embodiment, the sense region isconnected to the antisense region via a linker molecule, which can be apolynucleotide linker or a non-nucleotide linker.

In one embodiment, a siNA molecule of the invention comprises a senseregion and antisense region, wherein pyrimidine nucleotides in the senseregion comprise 2′-O-methyl pyrimidine nucleotides and purinenucleotides in the sense region comprise 2′-deoxy purine nucleotides. Inone embodiment, a siNA molecule of the invention comprises a senseregion and antisense region, wherein pyrimidine nucleotides present inthe sense region comprise 2′-deoxy-2′-fluoro pyrimidine nucleotides andwherein purine nucleotides present in the sense region comprise 2′-deoxypurine nucleotides.

In one embodiment, a siNA molecule of the invention comprises a senseregion and antisense region, wherein the pyrimidine nucleotides whenpresent in said antisense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides when present in said antisenseregion are 2′-O-methyl purine nucleotides.

In one embodiment, a siNA molecule of the invention comprises a senseregion and antisense region, wherein the pyrimidine nucleotides whenpresent in said antisense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and wherein the purine nucleotides when present in saidantisense region comprise 2′-deoxy-purine nucleotides.

In one embodiment, a siNA molecule of the invention comprises a senseregion and antisense region, wherein the sense region includes aterminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ends of the sense region. In another embodiment, the terminal cap moietyis an inverted deoxy abasic moiety.

In one embodiment, a siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a gene. Because many genescan share some degree of sequence homology with each other, siNAmolecules can be designed to target a class of genes (and associatedreceptor or ligand genes) or alternately specific genes by selectingsequences that are either shared amongst different gene targets oralternatively that are unique for a specific gene target. Therefore, inone embodiment, the siNA molecule can be designed to target conservedregions of a RNA sequence having homology between several genes so as totarget several genes or gene families (e.g., different gene isoforms,splice variants, mutant genes etc.) with one siNA molecule. In anotherembodiment, the siNA molecule can be designed to target a sequence thatis unique to a specific RNA sequence of a specific gene due to the highdegree of specificity that the siNA molecule requires to mediate RNAiactivity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplexes containing about 19 basepairs between oligonucleotides comprising about 19 to about 25 (e.g.,about 19, 20, 21, 22, 23, 24 or 25) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplexes withoverhanging ends of about 1 to about 3 (e.g., about 1, 2, or 3)nucleotides, for example, about 21-nucleotide duplexes with about 19base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs.

In one embodiment, the invention features one or morechemically-modified siNA constructs having specificity for nucleic acidmolecules that express or encode a protein sequence, such as RNA or DNAencoding a protein sequence. Non-limiting examples of such chemicalmodifications include without limitation phosphorothioateinternucleotide linkages, 2′-deoxyribonucleotides, 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, “acyclic” nucleotides, 5-C-methyl nucleotides, and terminalglyceryl and/or inverted deoxy abasic residue incorporation. Thesechemical modifications, when used in various siNA constructs, are shownto preserve RNAi activity in cells while at the same time, dramaticallyincreasing the serum stability of these compounds.

In one embodiment, a siNA molecule of the invention does not contain anyribonucleotides. In another embodiment, a siNA molecule of the inventioncomprises one or more ribonucleotides.

In one embodiment, the invention features the use of compounds orcompositions that inhibit the activity of double stranded RNA bindingproteins (dsRBPs, see for example Silhavy et al., 2003, Journal ofGeneral Virology, 84, 975-980). Non-limiting examples of compounds andcompositions that can be used to inhibit the activity of dsRBPs includebut are not limited to small molecules and nucleic acid aptamers thatbind to or interact with the dsRBPs and consequently reduce dsRBPactivity and/or siNA molecules that target nucleic acid sequencesencoding dsRBPs. The use of such compounds and compositions is expectedto improve the activity of siNA molecules in biological systems in whichdsRBPs can abrogate or suppress the efficacy of siNA mediated RNAinterference, such as where dsRBPs are expressed during viral infectionof a cell to escape RNAi surveillance. Therefore, the use of agents thatinhibit dsRBP activity is preferred in those instances where RNAinterference activity can be improved via the abrogation or suppressionof dsRBP activity. Such anti-dsRBP agents can be administered alone orcan be co-administered with siNA molecules of the invention, or can beused to pretreat cells or a subject before siNA administration. Inanother embodiment, anti-dsRBP agents are used to treat viral infection,such as HCV, HBV, or HIV infection with or without siNA molecules of theinvention.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a gene, wherein one of the strands of the double-stranded siNAmolecule comprises a nucleotide sequence that is complementary to anucleotide sequence of the gene or RNA encoded by the gene or a portionthereof, and wherein the second strand of the double-stranded siNAmolecule comprises a nucleotide sequence substantially similar to thenucleotide sequence of the gene or RNA encoded by the gene or a portionthereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a gene, wherein each strand of the siNA molecule comprises about 19to about 23 nucleotides, and wherein each strand comprises at leastabout 19 nucleotides that are complementary to the nucleotides of theother strand.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a gene, wherein the siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence of the gene or RNA encoded by the gene or a portion thereof,and wherein the siNA further comprises a sense region, wherein the senseregion comprises a nucleotide sequence substantially similar to thenucleotide sequence of the gene or RNA encoded by the gene or a portionthereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa target gene by mediating RNA interference (RNAi) process, wherein thesiNA molecule comprises no ribonucleotides and wherein each strand ofthe double-stranded siNA molecule comprises about 21 nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the replicationof a virus (e.g, as mammalian virus, plant virus, hepatitis C virus,human immunodeficiency virus, hepatitis B virus, herpes simplex virus,cytomegalovirus, human papilloma virus, respiratory syncytial virus, orinfluenza virus), wherein the siNA molecule does not require thepresence of a ribonucleotide within the siNA molecule for the inhibitionof replication of the virus and each strand of the double-stranded siNAmolecule comprises about 21 nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a gene, wherein the siNA molecule comprises a sense region and anantisense region and wherein the antisense region comprises a nucleotidesequence that is complementary to a nucleotide sequence or a portionthereof of RNA encoded by the gene and the sense region comprises anucleotide sequence that is complementary to the antisense region or aportion thereof, and wherein the purine nucleotides present in theantisense region comprise 2′-deoxy-purine nucleotides. In anotherembodiment, the purine nucleotides present in the antisense regioncomprise 2′-O-methyl purine nucleotides. In either of the aboveembodiments, the antisense region comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense region. In analternative embodiment, the antisense region comprises a glycerylmodification at the 3′ end of the antisense region. In anotherembodiment of any of the above described siNA molecules, any nucleotidespresent in a non-complementary region of the antisense strand (e.g.overhang region) are 2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a gene, wherein the siNA molecule is assembled from two separateoligonucleotide fragments each comprising 21 nucleotides, wherein onefragment comprises the sense region and the second fragment comprisesthe antisense region of the siNA molecule, and wherein about 19nucleotides of each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule andwherein at least two 3′ terminal nucleotides of each fragment of thesiNA molecule are not base-paired to the nucleotides of the otherfragment of the siNA molecule. In one embodiment, each of the two 3′terminal nucleotides of each fragment of the siNA molecule is a2′-deoxy-pyrimidine nucleotide, such as 2′-deoxy-thymidine. In anotherembodiment, all 21 nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule. In another embodiment, about 19 nucleotides of theantisense region are base-paired to the nucleotide sequence or a portionthereof of the RNA encoded by the gene. In another embodiment, 21nucleotides of the antisense region are base-paired to the nucleotidesequence or a portion thereof of the RNA encoded by the gene. In any ofthe above embodiments, the 5′-end of the fragment comprising saidantisense region can optionally include a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa RNA sequence (e.g., wherein said target RNA sequence is encoded by agene or a gene involved in a pathway of gene expression), wherein thesiNA molecule does not contain any ribonucleotides and wherein eachstrand of the double-stranded siNA molecule is about 21 nucleotideslong.

In one embodiment, the invention features a medicament comprising a siNAmolecule of the invention.

In one embodiment, the invention features an active ingredientcomprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to down-regulateexpression of a target gene, wherein the siNA molecule comprises one ormore chemical modifications and each strand of the double-stranded siNAis about 21 nucleotides long.

The invention features a double-stranded short interfering nucleic acid(siNA) molecule that inhibits expression of a gene, wherein one of thestrands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of a RNA encoded by the gene or a portion thereof, the otherstrand is a sense strand which comprises nucleotide sequence that iscomplementary to a nucleotide sequence of the antisense strand andwherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification. In oneembodiment, the nucleotide sequence of the antisense strand of thedouble-stranded siNA molecule is complementary to the nucleotidesequence of a RNA which encodes a protein or a portion thereof. In oneembodiment, each strand of the siNA molecule comprises about 19 to about29 (e.g., about 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29)nucleotides, and each strand comprises at least about 19 nucleotidesthat are complementary to the nucleotides of the other strand. In oneembodiment, the siNA molecule is assembled from two oligonucleotidefragments, wherein one fragment comprises the nucleotide sequence of theantisense strand of the siNA molecule and a second fragment comprisesnucleotide sequence of the sense region of the siNA molecule. In anotherembodiment, the sense strand is connected to the antisense strand via alinker molecule, such as a polynucleotide linker or a non-nucleotidelinker. In one embodiment, the pyrimidine nucleotides present in thesense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides and thepurine nucleotides present in the sense region are 2′-deoxy purinenucleotides. In another embodiment, the pyrimidine nucleotides presentin the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides andthe purine nucleotides present in the sense region are 2′-O-methylpurine nucleotides. In one embodiment, the sense strand comprises a3′-end and a 5′-end, wherein a terminal cap moiety (e.g., an inverteddeoxy abasic moiety) is present at the 5′-end, the 3′-end, or both ofthe 5′ and 3′ ends of the sense strand. In one embodiment, the antisensestrand comprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotidesand one or more 2′-O-methyl purine nucleotides. In one embodiment, thepyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-O-methyl purine nucleotides. Inone embodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group. In oneembodiment, the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a gene,wherein one of the strands of the double-stranded siNA molecule is anantisense strand which comprises nucleotide sequence that iscomplementary to nucleotide sequence of RNA encoded by a gene or aportion thereof, the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand and wherein a majority of the pyrimidinenucleotides present in the double-stranded siNA molecule comprises asugar modification, and wherein the nucleotide sequence of the antisensestrand is complementary to a nucleotide sequence of the 5′-untranslatedregion or a portion thereof of the RNA. In another embodiment, thenucleotide sequence of the antisense strand is complementary to anucleotide sequence of the RNA or a portion thereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of agene, wherein one of the strands of the double-stranded siNA molecule isan antisense strand which comprises nucleotide sequence that iscomplementary to nucleotide sequence of a RNA or a portion thereof, theother strand is a sense strand which comprises nucleotide sequence thatis complementary to a nucleotide sequence of the antisense strand andwherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification, andwherein each of the two strands of the siNA molecule comprises 21nucleotides. In one embodiment, about 19 nucleotides of each strand ofthe siNA molecule are base-paired to the complementary nucleotides ofthe other strand of the siNA molecule and at least two 3′ terminalnucleotides of each strand of the siNA molecule are not base-paired tothe nucleotides of the other strand of the siNA molecule. In oneembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule are 2′-deoxy-pyrimidines, such as 2′-deoxy-thymidine.In another embodiment, each strand of the siNA molecule is base-pairedto the complementary nucleotides of the other strand of the siNAmolecule. In one embodiment, about 19 nucleotides of the antisensestrand are base-paired to the nucleotide sequence of the RNA or aportion thereof. In another embodiment, 21 nucleotides of the antisensestrand are base-paired to the nucleotide sequence of the RNA or aportion thereof.

In one embodiment, the invention features a composition comprising asiNA molecule of the invention and a pharmaceutically acceptable carrieror diluent.

In one embodiment, the invention features a method of increasing thestability of a siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In another embodiment, all pyrimidine nucleotides present inthe siNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In anotherembodiment, the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridinenucleotides. In another embodiment, all uridine nucleotides present inthe siNA are 2′-deoxy-2′-fluoro uridine nucleotides. In anotherembodiment, all cytidine nucleotides present in the siNA are2′-deoxy-2′-fluoro cytidine nucleotides. In another embodiment, alladenosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroadenosine nucleotides. In another embodiment, all guanosine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro guanosine nucleotides. ThesiNA can further comprise at least one modified internucleotidiclinkage, such as phosphorothioate linkage. In another embodiment, the2′-deoxy-2′-fluoronucleotides are present at specifically selectedlocations in the siNA that are sensitive to cleavage by ribonucleases,such as locations having pyrimidine nucleotides.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits expressionof a gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of a RNA or a portion thereof,the other strand is a sense strand which comprises nucleotide sequencethat is complementary to a nucleotide sequence of the antisense strandand wherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule comprising a double-stranded structure thatdown-regulates expression of a target nucleic acid, wherein the siNAmolecule does not require a 2′-hydroxyl group containing ribonucleotide,each strand of the double-stranded structure of the siNA moleculecomprises about 21 nucleotides and the siNA molecule comprisesnucleotide sequence having complementarity to nucleotide sequence of thetarget nucleic acid or a portion thereof. The target nucleic acid can bean endogenous gene, an exogenous gene, a viral nucleic acid, or a RNA,such as a mammalian gene, plant gene, viral gene, fungal gene, bacterialgene, plant viral gene, or mammalian viral gene. Examples of mammalianviral gene include hepatitis C virus, human immunodeficiency virus,hepatitis B virus, herpes simplex virus, cytomegalovirus, humanpapilloma virus, respiratory syncytial virus, influenza virus, andsevere acute respiratory syndrome virus (SARS).

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region wherein the antisense region comprisesthe nucleotide sequence that is complementary to a nucleotide sequenceor a portion thereof of the target nucleic acid and the sense regioncomprises a nucleotide sequence that is complementary to nucleotidesequence of the antisense region or a portion thereof.

In one embodiment, a siNA molecule of the invention is assembled fromtwo separate oligonucleotide fragments wherein one fragment comprisesthe sense region and the second fragment comprises the antisense regionof the siNA molecule. The sense region can be connected to the antisenseregion via a linker molecule, such as a polynucleotide linker ornon-nucleotide linker. In another embodiment, each sense region andantisense region comprise about 21 nucleotides in length. In anotherembodiment, about 19 nucleotides of each fragment of the siNA moleculeare base-paired to the complementary nucleotides of the other fragmentof the siNA molecule and at least two 3′ terminal nucleotides of eachfragment of the siNA molecule are not base-paired to the nucleotides ofthe other fragment of the siNA molecule. In another embodiment, each ofthe two 3′ terminal nucleotides of each fragment of the siNA moleculeare 2′-deoxy-pyrimidines, such as the thymidine. In another embodiment,all 21 nucleotides of each fragment of the siNA molecule are base-pairedto the complementary nucleotides of the other fragment of the siNAmolecule. In another embodiment, about 19 nucleotides of the antisenseregion of the siNA molecule are base-paired to the nucleotide sequenceor a portion thereof of the target nucleic acid. In another embodiment,21 nucleotides of the antisense region of the siNA molecule arebase-paired to the nucleotide sequence or a portion thereof of thetarget nucleic acid. In another embodiment, the 5′-end of the fragmentcomprising the antisense region optionally includes a phosphate group.

In one embodiment, a siNA molecule of the invention comprises nucleotidesequence having complementarity to nucleotide sequence of RNA or aportion thereof encoded by the target nucleic acid or a portion thereof.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the pyrimidine nucleotides whenpresent in the sense region are 2′-O-methyl pyrimidine nucleotides andwherein the purine nucleotides when present in the sense region are2′-deoxy purine nucleotides.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the pyrimidine nucleotides whenpresent in the sense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and wherein the purine nucleotides when present in the senseregion are 2′-deoxy purine nucleotides.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the sense region includes aterminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ends. The cap moiety can be an inverted deoxy abasic moiety, an inverteddeoxy thymidine moiety, or a thymidine moiety.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the pyrimidine nucleotides whenpresent in the antisense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides when present in the antisenseregion are 2′-O-methyl purine nucleotides.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the pyrimidine nucleotides whenpresent in the antisense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and wherein the purine nucleotides when present in theantisense region comprise 2′-deoxy-purine nucleotides.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises aphosphate backbone modification at the 3′ end of the antisense region.The phosphate backbone modification can be a phosphorothioate.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises aglyceryl modification at the 3′ end of the antisense region.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein each of sense and the antisenseregions of the siNA molecule comprise about 21 nucleotides.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity in humans.

In any of the embodiments of siNA molecules described herein, theantisense region of a siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of a siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically-modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to an RNA or DNA sequence encoding a protein orpolypeptide and the sense region can comprise sequence complementary tothe antisense region. The siNA molecule can comprise two distinctstrands having complementary sense and antisense regions. The siNAmolecule can comprise a single strand having complementary sense andantisense regions.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbonemodified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring orchemically-modified, each X and Y is independently O, S, N, alkyl, orsubstituted alkyl, each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, or aralkyl, and wherein W,X, Y, and Z are optionally not all O. In another embodiment, a backbonemodification of the invention comprises a phosphonoacetate and/orthiophosphonoacetate internucleotide linkage (see for example Sheehan etal., 2003, Nucleic Acids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically-modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically-modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, a siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically-modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotideshaving Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be complementary or non-complementary to targetRNA or a non-nucleosidic base such as phenyl, naphthyl, 3-nitropyrrole,5-nitroindole, nebularine, pyridone, pyridinone, or any othernon-naturally occurring universal base that can be complementary ornon-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotide or non-nucleotide of Formula II at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides ornon-nucleotides of Formula II at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotides or non-nucleotides of Formula II at the 3′-end of the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotideshaving Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl, N-alkenyl,SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH, S-alkyl-OH,S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3, NH2,aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or group having Formula I or II; R9is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidic base such asadenine, guanine, uracil, cytosine, thymine, 2-aminoadenosine,5-methylcytosine, 2,6-diaminopurine, or any other non-naturallyoccurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA.

The chemically-modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotide or non-nucleotide of Formula III at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula III at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotide or non-nucleotide of Formula III at the 3′-end of the sensestrand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises a 5′-terminal phosphategroup having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, or alkylhalo oracetyl; and/or wherein W, X, Y and Z are not all O.

In one embodiment, the invention features a siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features a siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of a siNA molecule of the invention,for example a siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or morephosphorothioate, phosphonoacetate, and/or thiophosphonoacetateinternucleotide linkages. For example, in a non-limiting example, theinvention features a chemically-modified short interfering nucleic acid(siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in one siNA strand. In yet another embodiment,the invention features a chemically-modified short interfering nucleicacid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or morephosphorothioate internucleotide linkages in both siNA strands. Thephosphorothioate internucleotide linkages can be present in one or botholigonucleotide strands of the siNA duplex, for example in the sensestrand, the antisense strand, or both strands. The siNA molecules of theinvention can comprise one or more phosphorothioate, phosphonoacetate,and/or thiophosphonoacetate internucleotide linkages at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand, theantisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) consecutive phosphorothioateinternucleotide linkages at the 5′-end of the sense strand, theantisense strand, or both strands. In another non-limiting example, anexemplary siNA molecule of the invention can comprise one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands.

In one embodiment, the invention features a siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or about one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more, phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, ormore) universal base modified nucleotides, and optionally a terminal capmolecule at the 3-end, the 5′-end, or both of the 3′- and 5′-ends of thesense strand; and wherein the antisense strand comprises about 1 toabout 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal basemodified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 10 or more,specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more) universal base modified nucleotides, and optionally aterminal cap molecule at the 3′-end, the 5′-end, or both of the 3′- and5′-ends of the antisense strand. In another embodiment, one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidinenucleotides of the sense and/or antisense siNA strand arechemically-modified with 2′-deoxy, 2′-O-methyl and/or 2′-deoxy-2′-fluoronucleotides, with or without one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10 or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′ and 5′-ends, being present in the same or different strand.

In another embodiment, the invention features a siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the sense strand; and whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2-O-methyl, 2′-deoxy-2′-fluoro, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modifiednucleotides, and optionally a terminal cap molecule at the 3′-end, the5′-end, or both of the 3′- and 5′-ends of the antisense strand. Inanother embodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7,8, 9, 10 or more pyrimidine nucleotides of the sense and/or antisensesiNA strand are chemically-modified with 2′-deoxy, 2′-O-methyl and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5,specifically about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) canbe at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one orboth siNA sequence strands. In addition, the 2′-5′ internucleotidelinkage(s) can be present at various other positions within one or bothsiNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a pyrimidinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a purine nucleotidein one or both strands of the siNA molecule can comprise a 2′-5′internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is about 18 to about 27(e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) nucleotides inlength, wherein the duplex has about 18 to about 23 (e.g., about 18, 19,20, 21, 22, or 23) base pairs, and wherein the chemical modificationcomprises a structure having any of Formulae I-VII. For example, anexemplary chemically-modified siNA molecule of the invention comprises aduplex having two strands, one or both of which can bechemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, a siNA molecule of the invention comprises a single strandedhairpin structure, wherein the siNA is about 36 to about 70 (e.g., about36, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs, andwherein the siNA can include a chemical modification comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 42 to about 50 (e.g.,about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides that ischemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein the linear oligonucleotideforms a hairpin structure having about 19 base pairs and a 2-nucleotide3′-terminal nucleotide overhang. In another embodiment, a linear hairpinsiNA molecule of the invention contains a stem loop motif, wherein theloop portion of the siNA molecule is biodegradable. For example, alinear hairpin siNA molecule of the invention is designed such thatdegradation of the loop portion of the siNA molecule in vivo cangenerate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 23(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, or 23) base pairs and a 5′-terminal phosphate group thatcan be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In another embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 20 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20) base pairs, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically-modified siNA molecule of the invention comprises alinear oligonucleotide having about 25 to about 35 (e.g., about 25, 26,27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms an asymmetric hairpin structure having about 3 toabout 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17 or 18) base pairs and a 5′-terminal phosphate group that can bechemically modified as described herein (for example a 5′-terminalphosphate group having Formula IV). In another embodiment, an asymmetrichairpin siNA molecule of the invention contains a stem loop motif,wherein the loop portion of the siNA molecule is biodegradable. Inanother embodiment, an asymmetric hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 16 to about 25 (e.g., about 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides in length, wherein the sense region is about3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, or 18) nucleotides in length, wherein the sense region theantisense region have at least 3 complementary nucleotides, and whereinthe siNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises an asymmetric double stranded structure having separatepolynucleotide strands comprising sense and antisense regions, whereinthe antisense region is about 18 to about 22 (e.g., about 18, 19, 20,21, or 22) nucleotides in length and wherein the sense region is about 3to about 15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, a siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) basepairs, and wherein the siNA can include a chemical modification, whichcomprises a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a circular oligonucleotide having about 42 toabout 50 (e.g., about 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotidesthat is chemically-modified with a chemical modification having any ofFormulae I-VII or any combination thereof, wherein the circularoligonucleotide forms a dumbbell shaped structure having about 19 basepairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or group havingFormula I or II; R9 is O, S, CH2, S═O, CHF, or CF2, and either R3, R5,R8 or R13 serve as points of attachment to the siNA molecule of theinvention.

In another embodiment, a siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or a group havingFormula I, and R1, R2 or R3 serves as points of attachment to the siNAmolecule of the invention.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises Oand is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 22).

In another embodiment, a moiety having any of Formula V, VI or VII ofthe invention is at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of a siNA molecule of the invention. For example, a moietyhaving Formula V, VI or VII can be present at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the antisense strand, the sense strand, orboth antisense and sense strands of the siNA molecule. In addition, amoiety having Formula VII can be present at the 3′-end or the 5′-end ofa hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula V or VI is connected to the siNA construct in a 3-3′, 3-2′,2-3′, or 5-5′ configuration, such as at the 3′-end, the 5′-end, or bothof the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, the sense strand of a double stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.22) such as an inverted deoxyabasic moiety or inverted nucleotide, atthe 3′-end, 5′-end, or both 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically-modified siNA comprises a sense region, where any (e.g., oneor more or all) pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and where any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically-modified siNA comprises a sense region, where any (e.g., oneor more or all) pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and where any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides), wherein any nucleotides comprising a 3′-terminalnucleotide overhang that are present in said sense region are 2′-deoxynucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically-modified siNA comprises a sense region, where any (e.g., oneor more or all) pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and where any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically-modified siNA comprises a sense region, where any (e.g., oneor more or all) pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and where any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides), wherein any nucleotides comprising a3′-terminal nucleotide overhang that are present in said sense regionare 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically-modified siNA comprises an antisense region, where any (e.g.,one or more or all) pyrimidine nucleotides present in the antisenseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and wherein any (e.g., one or more or all)purine nucleotides present in the antisense region are 2′-O-methylpurine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methylpurine nucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically-modified siNA comprises an antisense region, where any (e.g.,one or more or all) pyrimidine nucleotides present in the antisenseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and wherein any (e.g., one or more or all)purine nucleotides present in the antisense region are 2′-O-methylpurine nucleotides (e.g., wherein all purine nucleotides are 2′-O-methylpurine nucleotides or alternately a plurality of purine nucleotides are2′-O-methyl purine nucleotides), wherein any nucleotides comprising a3′-terminal nucleotide overhang that are present in said antisenseregion are 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention, wherein thechemically-modified siNA comprises an antisense region, where any (e.g.,one or more or all) pyrimidine nucleotides present in the antisenseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and where any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-deoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) inside a cell or reconstituted invitro system comprising a sense region and an antisense region. In oneembodiment, the sense region comprises one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and one or more 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides). The sense regioncan comprise inverted deoxy abasic modifications that are optionallypresent at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of thesense region. The sense region can optionally further comprise a3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or4) 2′-deoxyribonucleotides. The antisense region comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more 2′-O-methyl purine nucleotides (e.g.,wherein all purine nucleotides are 2′-O-methyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). The antisense region can comprise a terminal capmodification, such as any modification described herein or shown in FIG.22, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence. The antisense regionoptionally further comprises a 3′-terminal nucleotide overhang havingabout 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides,wherein the overhang nucleotides can further comprise one or more (e.g.,1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limitingexamples of these chemically-modified siNAs are shown in FIGS. 18A-18Fand 19A-19F and Table IV herein.

In another embodiment of the chemically-modified short interferingnucleic acid comprising a sense region and an antisense region, thesense region comprises one or more 2′-deoxy-2′-fluoro pyrimidinenucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and one or more purine ribonucleotides (e.g., wherein all purinenucleotides are purine ribonucleotides or alternately a plurality ofpurine nucleotides are purine ribonucleotides). The sense region canalso comprise inverted deoxy abasic modifications that are optionallypresent at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of thesense region. The sense region optionally further comprises a3′-terminal overhang having about 1 to about 4 (e.g., about 1, 2, 3, or4) 2′-deoxyribonucleotides. The antisense region comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more 2′-O-methyl purine nucleotides (e.g.,wherein all purine nucleotides are 2′-O-methyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). The antisense region can also comprise a terminal capmodification, such as any modification described herein or shown in FIG.22, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence. The antisense regionoptionally further comprises a 3′-terminal nucleotide overhang havingabout 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides,wherein the overhang nucleotides can further comprise one or more (e.g.,1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limitingexamples of these chemically-modified siNAs are shown in FIGS. 18A-18Fand 19A-19F and Table IV herein.

In another embodiment of the chemically-modified short interferingnucleic acid comprising a sense region and an antisense region, thesense region comprises one or more 2′-deoxy-2′-fluoro pyrimidinenucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and one or more purine nucleotides selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides (e.g., wherein all purine nucleotides are selected from thegroup consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA)nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and2′-O-methyl nucleotides or alternately a plurality of purine nucleotidesare selected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides). The sense region cancomprise inverted deoxy abasic modifications that are optionally presentat the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of the senseregion. The sense region can optionally further comprise a 3′-terminaloverhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4)2′-deoxyribonucleotides. The antisense region comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more purine nucleotides selected from the groupconsisting of 2′-deoxy nucleotides, locked nucleic acid (LNA)nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and2′-O-methyl nucleotides (e.g., wherein all purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides or alternately aplurality of purine nucleotides are selected from the group consistingof 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides). The antisense can also comprise a terminal capmodification, such as any modification described herein or shown in FIG.22, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence. The antisense regionoptionally further comprises a 3′-terminal nucleotide overhang havingabout 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides,wherein the overhang nucleotides can further comprise one or more (e.g.,1, 2, 3, or 4) phosphorothioate internucleotide linkages.

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-0,4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′-methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, and 2′-O-methyl nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises a conjugate attached to thechemically-modified siNA molecule. The conjugate can be attached to thechemically-modified siNA molecule via a covalent attachment. In oneembodiment, the conjugate is attached to the chemically-modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically-modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically-modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically-modified siNA molecule, or any combination thereof. Inone embodiment, the conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically-modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically-modifiedsiNA molecule is a poly ethylene glycol, human serum albumin, or aligand for a cellular receptor that can mediate cellular uptake.Examples of specific conjugate molecules contemplated by the instantinvention that can be attached to chemically-modified siNA molecules aredescribed in Vargeese et al., U.S. Ser. No. 10/201,394, incorporated byreference herein. The type of conjugates used and the extent ofconjugation of siNA molecules of the invention can be evaluated forimproved pharmacokinetic profiles, bioavailability, and/or stability ofsiNA constructs while at the same time maintaining the ability of thesiNA to mediate RNAi activity. As such, one skilled in the art canscreen siNA constructs that are modified with various conjugates todetermine whether the siNA conjugate complex possesses improvedproperties while maintaining the ability to mediate RNAi, for example inanimal models as are generally known in the art.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) inside a cell or reconstituted invitro system, wherein the chemically-modified siNA comprises a senseregion, where one or more pyrimidine nucleotides present in the senseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and where one or more purine nucleotidespresent in the sense region are 2′-deoxy purine nucleotides (e.g.,wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality of purine nucleotides are 2′-deoxy purinenucleotides), and inverted deoxy abasic modifications that areoptionally present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the sense region, the sense region optionally furthercomprising a 3′-terminal overhang having about 1 to about 4 (e.g., about1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein thechemically-modified short interfering nucleic acid molecule comprises anantisense region, where one or more pyrimidine nucleotides present inthe antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides(e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides or alternately a plurality of pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and whereinone or more purine nucleotides present in the antisense region are2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are2′-O-methyl purine nucleotides or alternately a plurality of purinenucleotides are 2′-O-methyl purine nucleotides), and a terminal capmodification, such as any modification described herein or shown in FIG.22, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence, the antisense regionoptionally further comprising a 3′-terminal nucleotide overhang havingabout 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides,wherein the overhang nucleotides can further comprise one or more (e.g.,1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limitingexamples of these chemically-modified siNAs are shown in FIGS. 18A-18Fand 19A-19F and Table IV herein.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) inside a cell or reconstituted invitro system, wherein the chemically-modified siNA comprises a senseregion, where one or more pyrimidine nucleotides present in the senseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and where one or more purine nucleotidespresent in the sense region are 2′-O-methyl purine nucleotides (e.g.,wherein all purine nucleotides are 2′-O-methyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides), and inverted deoxy abasic modifications that areoptionally present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the sense region, the sense region optionally furthercomprising a 3′-terminal overhang having about 1 to about 4 (e.g., about1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein thechemically-modified short interfering nucleic acid molecule comprises anantisense region, where one or more pyrimidine nucleotides present inthe antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides(e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides or alternately a plurality of pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and whereinone or more purine nucleotides present in the antisense region are2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are2′-O-methyl purine nucleotides or alternately a plurality of purinenucleotides are 2′-O-methyl purine nucleotides), and a terminal capmodification, such as any modification described herein or shown in FIG.22, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence, the antisense regionoptionally further comprising a 3′-terminal nucleotide overhang havingabout 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides,wherein the overhang nucleotides can further comprise one or more (e.g.,1, 2, 3, or 4) phosphorothioate internucleotide linkages. Non-limitingexamples of these chemically-modified siNAs are shown in FIGS. 18A-18Fand 19A-19F and Table IV herein.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) inside a cell or reconstituted invitro system, wherein the siNA comprises a sense region, where one ormore pyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and where one or more purine nucleotides present in thesense region are purine ribonucleotides (e.g., wherein all purinenucleotides are purine ribonucleotides or alternately a plurality ofpurine nucleotides are purine ribonucleotides), and inverted deoxyabasic modifications that are optionally present at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the sense region, the senseregion optionally further comprising a 3′-terminal overhang having about1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxyribonucleotides; andwherein the siNA comprises an antisense region, where one or morepyrimidine nucleotides present in the antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), anda terminal cap modification, such as any modification described hereinor shown in FIG. 22, that is optionally present at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the antisense sequence, theantisense region optionally further comprising a 3′-terminal nucleotideoverhang having about 1 to about 4 (e.g., about 1, 2, 3, or 4)2′-deoxynucleotides, wherein the overhang nucleotides can furthercomprise one or more (e.g., 1, 2, 3, or 4) phosphorothioateinternucleotide linkages. Non-limiting examples of thesechemically-modified siNAs are shown in FIGS. 18A-18F and 19A-19F andTable IV herein.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention capable ofmediating RNA interference (RNAi) inside a cell or reconstituted invitro system, wherein the chemically-modified siNA comprises a senseregion, where one or more pyrimidine nucleotides present in the senseregion are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides oralternately a plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and for example where one or more purinenucleotides present in the sense region are selected from the groupconsisting of 2′-deoxy nucleotides, locked nucleic acid (LNA)nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and2′-O-methyl nucleotides (e.g., wherein all purine nucleotides areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides or alternately aplurality of purine nucleotides are selected from the group consistingof 2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, and 2′-O-methylnucleotides), and wherein inverted deoxy abasic modifications areoptionally present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the sense region, the sense region optionally furthercomprising a 3′-terminal overhang having about 1 to about 4 (e.g., about1, 2, 3, or 4) 2′-deoxyribonucleotides; and wherein thechemically-modified short interfering nucleic acid molecule comprises anantisense region, where one or more pyrimidine nucleotides present inthe antisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides(e.g., wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides or alternately a plurality of pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides), and whereinone or more purine nucleotides present in the antisense region areselected from the group consisting of 2′-deoxy nucleotides, lockednucleic acid (LNA) nucleotides, 2′-methoxyethyl nucleotides,4′-thionucleotides, and 2′-O-methyl nucleotides (e.g., wherein allpurine nucleotides are selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, and 2′-O-methyl nucleotides oralternately a plurality of purine nucleotides are selected from thegroup consisting of 2′-deoxy nucleotides, locked nucleic acid (LNA)nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides, and2′-O-methyl nucleotides), and a terminal cap modification, such as anymodification described herein or shown in FIG. 22, that is optionallypresent at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of theantisense sequence, the antisense region optionally further comprising a3′-terminal nucleotide overhang having about 1 to about 4 (e.g., about1, 2, 3, or 4) 2′-deoxynucleotides, wherein the overhang nucleotides canfurther comprise one or more (e.g., 1, 2, 3, or 4) phosphorothioateinternucleotide linkages.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotidelinker of the invention can be a linker of ≥2 nucleotides in length, forexample 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In anotherembodiment, the nucleotide linker can be a nucleic acid aptamer. By“aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that comprises a sequence recognizedby the target molecule in its natural setting. Alternately, an aptamercan be a nucleic acid molecule that binds to a target molecule where thetarget molecule does not naturally bind to a nucleic acid. The targetmolecule can be any molecule of interest. For example, the aptamer canbe used to bind to a ligand-binding domain of a protein, therebypreventing interaction of the naturally occurring ligand with theprotein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art (see, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.) In yet anotherembodiment, a non-nucleotide linker of the invention comprises abasicnucleotide, polyether, polyamine, polyamide, peptide, carbohydrate,lipid, polyhydrocarbon, or other polymeric compounds (e.g. polyethyleneglycols such as those having between 2 and 100 ethylene glycol units).Specific examples include those described by Seela and Kaiser, NucleicAcids Res. 1990, 18:6353 and Nucleic Acids Res. 1987, 15:3113; Cload andSchepartz, J. Am. Chem. Soc. 1991, 113:6324; Richardson and Schepartz,J. Am. Chem. Soc. 1991, 113:5109; Ma et al., Nucleic Acids Res. 1993,21:2585 and Biochemistry 1993, 32:1751; Durand et al., Nucleic AcidsRes. 1990, 18:6353; McCurdy et al., Nucleosides & Nucleotides 1991,10:287; Jschke et al., Tetrahedron Lett. 1993, 34:301; Ono et al.,Biochemistry 1991, 30:9914; Arnold et al., International Publication No.WO 89/02439; Usman et al., International Publication No. WO 95/06731;Dudycz et al., International Publication No. WO 95/11910 and Ferentz andVerdine, J. Am. Chem. Soc. 1991, 113:4000, all hereby incorporated byreference herein. A “non-nucleotide” further means any group or compoundthat can be incorporated into a nucleic acid chain in the place of oneor more nucleotide units, including either sugar and/or phosphatesubstitutions, and allows the remaining bases to exhibit their enzymaticactivity. The group or compound can be abasic in that it does notcontain a commonly recognized nucleotide base, such as adenosine,guanine, cytosine, uracil or thymine, for example at the C1 position ofthe sugar.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) against a target gene inside a cell or reconstitutedin vitro system, wherein the chemical modification comprises a conjugatecovalently attached to the chemically-modified siNA molecule.Non-limiting examples of conjugates contemplated by the inventioninclude conjugates and ligands described in Vargeese et al., U.S. Ser.No. 10/427,160, filed Apr. 30, 2003, incorporated by reference herein inits entirety, including the drawings. In another embodiment, theconjugate is covalently attached to the chemically-modified siNAmolecule via a biodegradable linker. In one embodiment, the conjugatemolecule is attached at the 3′-end of either the sense strand, theantisense strand, or both strands of the chemically-modified siNAmolecule. In another embodiment, the conjugate molecule is attached atthe 5′-end of either the sense strand, the antisense strand, or bothstrands of the chemically-modified siNA molecule. In yet anotherembodiment, the conjugate molecule is attached both the 3′-end and5′-end of either the sense strand, the antisense strand, or both strandsof the chemically-modified siNA molecule, or any combination thereof. Inone embodiment, a conjugate molecule of the invention comprises amolecule that facilitates delivery of a chemically-modified siNAmolecule into a biological system, such as a cell. In anotherembodiment, the conjugate molecule attached to the chemically-modifiedsiNA molecule is a polyethylene glycol, human serum albumin, or a ligandfor a cellular receptor that can mediate cellular uptake. Examples ofspecific conjugate molecules contemplated by the instant invention thatcan be attached to chemically-modified siNA molecules are described inVargeese et al., U.S. Ser. No. 10/201,394, incorporated by referenceherein. The type of conjugates used and the extent of conjugation ofsiNA molecules of the invention can be evaluated for improvedpharmacokinetic profiles, bioavailability, and/or stability of siNAconstructs while at the same time maintaining the ability of the siNA tomediate RNAi activity. As such, one skilled in the art can screen siNAconstructs that are modified with various conjugates to determinewhether the siNA conjugate complex possesses improved properties whilemaintaining the ability to mediate RNAi, for example in animal models asare generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, a siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, a siNA molecule can beassembled from a single oligonucleotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, the invention features a siNA molecule that does notrequire the presence of a 2′-OH group (ribonucleotide) to be presentwithin the siNA molecule to support RNA interference.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system, wherein the siNA molecule comprises a single strandedpolynucleotide having complementarity to a target nucleic acid sequence.In another embodiment, the single stranded siNA molecule of theinvention comprises a 5′-terminal phosphate group. In anotherembodiment, the single stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group and a 3′-terminal phosphate group (e.g., a2′,3′-cyclic phosphate). In another embodiment, the single stranded siNAmolecule of the invention comprises about 19 to about 29 (e.g., about19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29) nucleotides. In yetanother embodiment, the single stranded siNA molecule of the inventioncomprises one or more chemically modified nucleotides or non-nucleotidesdescribed herein. For example, all the positions within the siNAmolecule can include chemically-modified nucleotides such as nucleotideshaving any of Formulae I-VII, or any combination thereof to the extentthat the ability of the siNA molecule to support RNAi activity in a cellis maintained.

In one embodiment, the single stranded siNA molecule havingcomplementarity to a target nucleic acid sequence comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and one or more 2′-O-methyl purine nucleotides (e.g.,wherein all purine nucleotides are 2′-O-methyl purine nucleotides oralternately a plurality of purine nucleotides are 2′-O-methyl purinenucleotides). In another embodiment, the single stranded siNA moleculecomprises one or more 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality of pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides), and one or more 2′-deoxypurine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxypurine nucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides). In another embodiment, the single strandedsiNA molecule comprises one or more 2′-deoxy-2′-fluoro pyrimidinenucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),wherein any purine nucleotides present in the antisense region arelocked nucleic acid (LNA) nucleotides (e.g., wherein all purinenucleotides are LNA nucleotides or alternately a plurality of purinenucleotides are LNA nucleotides). In another embodiment, the singlestranded siNA molecule comprises one or more 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides),and one or more 2′-methoxyethyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-methoxyethyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-methoxyethyl purinenucleotides), the single stranded siNA can comprise a terminal capmodification, such as any modification described herein or shown in FIG.22, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence. The single stranded siNAoptionally further comprises about 1 to about 4 (e.g., about 1, 2, 3, or4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule,wherein the terminal nucleotides can further comprise one or more (e.g.,1, 2, 3, or 4) phosphorothioate internucleotide linkages. The singlestranded siNA optionally further comprises a terminal phosphate group,such as a 5′-terminal phosphate group.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system, wherein the siNA molecule comprises a single strandedpolynucleotide having complementarity to a target nucleic acid sequence,and wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in theantisense region are 2′-O-methyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-O-methyl purine nucleotides or alternately aplurality of purine nucleotides are 2′-O-methyl purine nucleotides), anda terminal cap modification, such as any modification described hereinor shown in FIG. 22, that is optionally present at the 3′-end, the5′-end, or both of the 3′ and 5′-ends of the antisense sequence. ThesiNA optionally further comprises about 1 to about 4 or more (e.g.,about 1, 2, 3, 4 or more) terminal 2′-deoxynucleotides at the 3′-end ofthe siNA molecule, wherein the terminal nucleotides can further compriseone or more (e.g., 1, 2, 3, 4 or more) phosphorothioate,phosphonoacetate, and/or thiophosphonoacetate internucleotide linkages,and wherein the siNA optionally further comprises a terminal phosphategroup, such as a 5′-terminal phosphate group. In any of theseembodiments, any purine nucleotides present in the antisense region arealternatively 2′-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a pluralityof purine nucleotides are 2′-deoxy purine nucleotides). Also, in any ofthese embodiments, any purine nucleotides present in the siNA (i.e.,purine nucleotides present in the sense and/or antisense region) canalternatively be locked nucleic acid (LNA) nucleotides (e.g., whereinall purine nucleotides are LNA nucleotides or alternately a plurality ofpurine nucleotides are LNA nucleotides). Also, in any of theseembodiments, any purine nucleotides present in the siNA arealternatively 2′-methoxyethyl purine nucleotides (e.g., wherein allpurine nucleotides are 2′-methoxyethyl purine nucleotides or alternatelya plurality of purine nucleotides are 2′-methoxyethyl purinenucleotides).

In another embodiment, any modified nucleotides present in the singlestranded siNA molecules of the invention comprise modified nucleotideshaving properties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the single stranded siNA molecules ofthe invention are preferably resistant to nuclease degradation while atthe same time maintaining the capacity to mediate RNAi

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system, wherein the siNA molecule comprises a single strandedpolynucleotide having complementarity to a target nucleic acid sequence,and wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in the siNA are2′-O-methyl purine nucleotides (e.g., wherein all purine nucleotides are2′-O-methyl purine nucleotides or alternately a plurality of purinenucleotides are 2′-O-methyl purine nucleotides), and a terminal capmodification, such as any modification described herein or shown in FIG.22, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence, the siNA optionally furthercomprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein theterminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or4) phosphorothioate internucleotide linkages, and wherein the siNAoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system, wherein the siNA molecule comprises a single strandedpolynucleotide having complementarity to a target nucleic acid sequence,and wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in the siNA are2′-deoxy purine nucleotides (e.g., wherein all purine nucleotides are2′-deoxy purine nucleotides or alternately a plurality of purinenucleotides are 2′-deoxy purine nucleotides), and a terminal capmodification, such as any modification described herein or shown in FIG.22, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence, the siNA optionally furthercomprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein theterminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or4) phosphorothioate internucleotide linkages, and wherein the siNAoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system, wherein the siNA molecule comprises a single strandedpolynucleotide having complementarity to a target nucleic acid sequence,and wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in the siNA arelocked nucleic acid (LNA) nucleotides (e.g., wherein all purinenucleotides are LNA nucleotides or alternately a plurality of purinenucleotides are LNA nucleotides), and a terminal cap modification, suchas any modification described herein or shown in FIG. 22, that isoptionally present at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of the antisense sequence, the siNA optionally furthercomprising about 1 to about 4 (e.g., about 1, 2, 3, or 4) terminal2′-deoxynucleotides at the 3′-end of the siNA molecule, wherein theterminal nucleotides can further comprise one or more (e.g., 1, 2, 3, or4) phosphorothioate internucleotide linkages, and wherein the siNAoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system, wherein the siNA molecule comprises a single strandedpolynucleotide having complementarity to a target nucleic acid sequence,and wherein one or more pyrimidine nucleotides present in the siNA are2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g., wherein all pyrimidinenucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides or alternatelya plurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides), and wherein any purine nucleotides present in the siNA are2′-methoxyethyl purine nucleotides (e.g., wherein all purine nucleotidesare 2′-methoxyethyl purine nucleotides or alternately a plurality ofpurine nucleotides are 2′-methoxyethyl purine nucleotides), and aterminal cap modification, such as any modification described herein orshown in FIG. 22, that is optionally present at the 3′-end, the 5′-end,or both of the 3′ and 5′-ends of the antisense sequence, the siNAoptionally further comprising about 1 to about 4 (e.g., about 1, 2, 3,or 4) terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule,wherein the terminal nucleotides can further comprise one or more (e.g.,1, 2, 3, or 4) phosphorothioate internucleotide linkages, and whereinthe siNA optionally further comprises a terminal phosphate group, suchas a 5′-terminal phosphate group.

In another embodiment, any modified nucleotides present in the singlestranded siNA molecules of the invention comprise modified nucleotideshaving properties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984). As such, chemicallymodified nucleotides present in the single stranded siNA molecules ofthe invention are preferably resistant to nuclease degradation while atthe same time maintaining the capacity to mediate RNAi.

In one embodiment, the invention features a method for modulating theexpression of a gene within a cell comprising: (a) synthesizing a siNAmolecule of the invention, which can be chemically-modified, wherein oneof the siNA strands comprises a sequence complementary to RNA of thegene; and (b) introducing the siNA molecule into a cell under conditionssuitable to modulate the expression of the gene in the cell.

In one embodiment, the invention features a method for modulating theexpression of a gene within a cell comprising: (a) synthesizing a siNAmolecule of the invention, which can be chemically-modified, wherein oneof the siNA strands comprises a sequence complementary to RNA of thegene and wherein the sense strand sequence of the siNA comprises asequence substantially similar to the sequence of the target RNA; and(b) introducing the siNA molecule into a cell under conditions suitableto modulate the expression of the gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the genes; and (b) introducing the siNAmolecules into a cell under conditions suitable to modulate theexpression of the genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one gene within a cell comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the gene and wherein the sense strandsequence of the siNA comprises a sequence substantially similar to thesequence of the target RNA; and (b) introducing the siNA molecules intoa cell under conditions suitable to modulate the expression of the genesin the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g. using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients. Non-limiting examples of exvivo applications include use in organ/tissue transplant, tissuegrafting, or treatment of pulmonary disease (e.g., restenosis) orprevent neointimal hyperplasia and atherosclerosis in vein grafts. Suchex vivo applications may also used to treat conditions associated withcoronary and peripheral bypass graft failure, for example, such methodscan be used in conjunction with peripheral vascular bypass graft surgeryand coronary artery bypass graft surgery. Additional applicationsinclude transplants to treat CNS lesions or injury, including use intreatment of neurodegenerative conditions such as Alzheimer's disease,Parkinson's Disease, Epilepsy, Dementia, Huntington's disease, oramyotrophic lateral sclerosis (ALS).

In one embodiment, the invention features a method of modulating theexpression of a gene in a tissue explant comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the gene; and (b) introducing the siNA molecule into a cell ofthe tissue explant derived from a particular organism under conditionssuitable to modulate the expression of the gene in the tissue explant.In another embodiment, the method further comprises introducing thetissue explant back into the organism the tissue was derived from orinto another organism under conditions suitable to modulate theexpression of the gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a gene in a tissue explant comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified,wherein one of the siNA strands comprises a sequence complementary toRNA of the gene and wherein the sense strand sequence of the siNAcomprises a sequence substantially similar to the sequence of the targetRNA; and (b) introducing the siNA molecule into a cell of the tissueexplant derived from a particular organism under conditions suitable tomodulate the expression of the gene in the tissue explant. In anotherembodiment, the method further comprises introducing the tissue explantback into the organism the tissue was derived from or into anotherorganism under conditions suitable to modulate the expression of thegene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one gene in a tissue explant comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the genes; and (b) introducing the siNAmolecules into a cell of the tissue explant derived from a particularorganism under conditions suitable to modulate the expression of thegenes in the tissue explant. In another embodiment, the method furthercomprises introducing the tissue explant back into the organism thetissue was derived from or into another organism under conditionssuitable to modulate the expression of the genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a gene in an organism comprising: (a) synthesizing a siNAmolecule of the invention, which can be chemically-modified, wherein oneof the siNA strands comprises a sequence complementary to RNA of thegene; and (b) introducing the siNA molecule into the organism underconditions suitable to modulate the expression of the gene in theorganism.

In another embodiment, the invention features a method of modulating theexpression of more than one gene in an organism comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the genes; and (b) introducing the siNAmolecules into the organism under conditions suitable to modulate theexpression of the genes in the organism.

In one embodiment, the invention features a method for modulating theexpression of a gene within a cell comprising: (a) synthesizing a siNAmolecule of the invention, which can be chemically-modified, wherein thesiNA comprises a single stranded sequence having complementarity to RNAof the gene; and (b) introducing the siNA molecule into a cell underconditions suitable to modulate the expression of the gene in the cell.

In one embodiment, the invention features a method of modulating theexpression of a target gene in an tissue or organ comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the target gene; and (b)introducing the siNA molecule into the tissue or organ under conditionssuitable to modulate the expression of the target gene in the organism.In another embodiment, the tissue is ocular tissue and the organ is theeye. In another embodiment, the tissue comprises hepatocytes and/orhepatic tissue and the organ is the liver.

In another embodiment, the invention features a method for modulatingthe expression of more than one gene within a cell comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the gene; and (b) contactingthe siNA molecule with a cell in vitro or in vivo under conditionssuitable to modulate the expression of the genes in the cell.

In one embodiment, the invention features a method of modulating theexpression of a gene in a tissue explant comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified,wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the gene; and (b) contacting the siNA moleculewith a cell of the tissue explant derived from a particular organismunder conditions suitable to modulate the expression of the gene in thetissue explant. In another embodiment, the method further comprisesintroducing the tissue explant back into the organism the tissue wasderived from or into another organism under conditions suitable tomodulate the expression of the gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one gene in a tissue explant comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the gene; and (b) introducingthe siNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate the expressionof the genes in the tissue explant. In another embodiment, the methodfurther comprises introducing the tissue explant back into the organismthe tissue was derived from or into another organism under conditionssuitable to modulate the expression of the genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a gene in an organism comprising: (a) synthesizing a siNAmolecule of the invention, which can be chemically-modified, wherein thesiNA comprises a single stranded sequence having complementarity to RNAof the gene; and (b) introducing the siNA molecule into the organismunder conditions suitable to modulate the expression of the gene in theorganism.

In another embodiment, the invention features a method of modulating theexpression of more than one gene in an organism comprising: (a)synthesizing siNA molecules of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the gene; and (b) introducingthe siNA molecules into the organism under conditions suitable tomodulate the expression of the genes in the organism.

In one embodiment, the invention features a method of modulating theexpression of a gene in an organism comprising contacting the organismwith a siNA molecule of the invention under conditions suitable tomodulate the expression of the gene in the organism.

In another embodiment, the invention features a method of modulating theexpression of more than one gene in an organism comprising contactingthe organism with one or more siNA molecules of the invention underconditions suitable to modulate the expression of the genes in theorganism.

The siNA molecules of the invention can be designed to down regulate orinhibit target gene expression through RNAi targeting of a variety ofRNA molecules. In one embodiment, the siNA molecules of the inventionare used to target various RNAs corresponding to a target gene.Non-limiting examples of such RNAs include messenger RNA (mRNA),alternate RNA splice variants of target gene(s), post-transcriptionallymodified RNA of target gene(s), pre-mRNA of target gene(s), and/or RNAtemplates. If alternate splicing produces a family of transcripts thatare distinguished by usage of appropriate exons, the instant inventioncan be used to inhibit gene expression through the appropriate exons tospecifically inhibit or to distinguish among the functions of genefamily members. For example, a protein that contains an alternativelyspliced transmembrane domain can be expressed in both membrane bound andsecreted forms. Use of the invention to target the exon containing thetransmembrane domain can be used to determine the functionalconsequences of pharmaceutical targeting of membrane bound as opposed tothe secreted form of the protein. Non-limiting examples of applicationsof the invention relating to targeting these RNA molecules includetherapeutic pharmaceutical applications, pharmaceutical discoveryapplications, molecular diagnostic and gene function applications, andgene mapping, for example using single nucleotide polymorphism mappingwith siNA molecules of the invention. Such applications can beimplemented using known gene sequences or from partial sequencesavailable from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies. As such, siNA molecules targeting multiple gene targets canprovide increased therapeutic effect. In addition, siNA can be used tocharacterize pathways of gene function in a variety of applications. Forexample, the present invention can be used to inhibit the activity oftarget gene(s) in a pathway to determine the function of uncharacterizedgene(s) in gene function analysis, mRNA function analysis, ortranslational analysis. The invention can be used to determine potentialtarget gene pathways involved in various diseases and conditions towardpharmaceutical development. The invention can be used to understandpathways of gene expression involved in, for example, in development,such as prenatal development and postnatal development, and/or theprogression and/or maintenance of cancer, infectious disease,autoimmunity, inflammation, endocrine disorders, renal disease,pulmonary disease, cardiovascular disease, birth defects, ageing, anyother disease or condition related to gene expression.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down-regulate or inhibit the expression of gene(s) that encodeRNA referred to by Genbank Accession, for example genes encoding RNAsequence(s) referred to herein by Genbank Accession number.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 19 to about 25 (e.g., about 19, 20, 21,22, 23, 24, or 25) nucleotides in length. In one embodiment, the assaycan comprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. In another embodiment, fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by cellular expression in in vivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4^(N), where N represents thenumber of base paired nucleotides in each of the siNA construct strands(eg. for a siNA construct having 21 nucleotide sense and antisensestrands with 19 base pairs, the complexity would be 4¹⁹); and (b)assaying the siNA constructs of (a) above, under conditions suitable todetermine RNAi target sites within the target RNA sequence. In anotherembodiment, the siNA molecules of (a) have strands of a fixed length,for example about 23 nucleotides in length. In yet another embodiment,the siNA molecules of (a) are of differing length, for example havingstrands of about 19 to about 25 (e.g., about 19, 20, 21, 22, 23, 24, or25) nucleotides in length. In one embodiment, the assay can comprise areconstituted in vitro siNA assay as described in Example 7 herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. In another embodiment, fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. In another embodiment, the target RNA sequence can beobtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 19 to about25 (e.g., about 19, 20, 21, 22, 23, 24, or 25) nucleotides in length. Inone embodiment, the assay can comprise a reconstituted in vitro siNAassay as described herein. In another embodiment, the assay can comprisea cell culture system in which target RNA is expressed. Fragments oftarget RNA are analyzed for detectable levels of cleavage, for exampleby gel electrophoresis, northern blot analysis, or RNAse protectionassays, to determine the most suitable target site(s) within the targetRNA sequence. The target RNA sequence can be obtained as is known in theart, for example, by cloning and/or transcription for in vitro systems,and by expression in in vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by a siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising asiNA molecule of the invention, which can be chemically-modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically-modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease or condition in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thediagnosis of the disease or condition in the subject. In anotherembodiment, the invention features a method for treating or preventing adisease or condition in a subject, comprising administering to thesubject a composition of the invention under conditions suitable for thetreatment or prevention of the disease or condition in the subject,alone or in conjunction with one or more other therapeutic compounds. Inyet another embodiment, the invention features a method for reducing orpreventing tissue rejection in a subject comprising administering to thesubject a composition of the invention under conditions suitable for thereduction or prevention of tissue rejection in the subject.

In another embodiment, the invention features a method for validating agene target, comprising: (a) synthesizing a siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a target gene; (b)introducing the siNA molecule into a cell, tissue, or organism underconditions suitable for modulating expression of the target gene in thecell, tissue, or organism; and (c) determining the function of the geneby assaying for any phenotypic change in the cell, tissue, or organism.

In another embodiment, the invention features a method for validating atarget gene comprising: (a) synthesizing a siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a target gene; (b)introducing the siNA molecule into a biological system under conditionssuitable for modulating expression of the target gene in the biologicalsystem; and (c) determining the function of the gene by assaying for anyphenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human,animal, plant, insect, bacterial, viral or other sources, wherein thesystem comprises the components required for RNAi activity. The term“biological system” includes, for example, a cell, tissue, or organism,or extract thereof. The term biological system also includesreconstituted RNAi systems that can be used in an in vitro setting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of a target gene in biological system,including, for example, in a cell, tissue, or organism. In anotherembodiment, the invention features a kit containing more than one siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of more than one target gene in abiological system, including, for example, in a cell, tissue, ororganism.

In one embodiment, the invention features a kit containing a siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of a target gene in a biological system.In another embodiment, the invention features a kit containing more thanone siNA molecule of the invention, which can be chemically-modified,that can be used to modulate the expression of more than one target genein a biological system.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically-modified. Inanother embodiment, the cell containing a siNA molecule of the inventionis a mammalian cell. In yet another embodiment, the cell containing asiNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention,which can be chemically-modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing asiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for example,under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizinga siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi in a cell or reconstituted system, wherein the siNA constructcomprises one or more chemical modifications, for example, one or morechemical modifications having any of Formulae I-VII or any combinationthereof that increases the nuclease resistance of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into a siNA molecule, and (b) assaying the siNA molecule of step(a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target gene, wherein the siNA construct comprises one ormore chemical modifications described herein that modulates the bindingaffinity between the sense and antisense strands of the siNA construct.

In one embodiment, the binding affinity between the sense and antisensestrands of the siNA construct is modulated to increase the activity ofthe siNA molecule with regard to the ability of the siNA to mediate RNAinterference. In another embodiment the binding affinity between thesense and antisense strands of the siNA construct is decreased. Thebinding affinity between the sense and antisense strands of the siNAconstruct can be decreased by introducing one or more chemicallymodified nucleotides in the siNA sequence that disrupts the duplexstability of the siNA (e.g., lowers the Tm of the duplex). The bindingaffinity between the sense and antisense strands of the siNA constructcan be decreased by introducing one or more nucleotides in the siNAsequence that do not form Watson-Crick base pairs. The binding affinitybetween the sense and antisense strands of the siNA construct can bedecreased by introducing one or more wobble base pairs in the siNAsequence. The binding affinity between the sense and antisense strandsof the siNA construct can be decreased by modifying the nucleobasecomposition of the siNA, such as by altering the G-C content of the siNAsequence (e.g., decreasing the number of G-C base pairs in the siNAsequence). These modifications and alterations in sequence can beintroduced selectively at pre-determined positions of the siNA sequenceto increase siNA mediated RNAi activity. For example, such modificationsand sequence alterations can be introduced to disrupt siNA duplexstability between the 5′-end of the antisense strand and the 3′-end ofthe sense strand, the 3′-end of the antisense strand and the 5′-end ofthe sense strand, or alternately the middle of the siNA duplex. Inanother embodiment, siNA molecules are screened for optimized RNAiactivity by introducing such modifications and sequence alterationseither by rational design based upon observed rules or trends inincreasing siNA activity, or randomly via combinatorial selectionprocesses that cover either partial or complete sequence space of thesiNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoa siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi in a cell or reconstituted system, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the binding affinity between the antisense strand of the siNAconstruct and a complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi in a cell or reconstituted system, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the binding affinity between the antisense strand of the siNAconstruct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi in a cell or reconstituted system, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulate the polymerase activity of a cellular polymerase capable ofgenerating additional endogenous siNA molecules having sequence homologyto the chemically-modified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically-modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically-modified siNAmolecule. In one embodiment, the invention features chemically-modifiedsiNA constructs that mediate RNAi in a cell or reconstituted system,wherein the chemical modifications do not significantly effect theinteraction of siNA with a target RNA molecule, DNA molecule and/orproteins or other factors that are essential for RNAi in a manner thatwould decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity comprising (a) introducingnucleotides having any of Formula I-VII or any combination thereof intoa siNA molecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improved RNAiactivity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against a targetRNA comprising (a) introducing nucleotides having any of Formula I-VIIor any combination thereof into a siNA molecule, and (b) assaying thesiNA molecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against a DNAtarget comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi activity against the DNA target,such as a gene, chromosome, or portion thereof.

In one embodiment, the invention features siNA constructs that mediateRNAi in a cell or reconstituted system, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the cellular uptake of the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules against a target gene with improved cellular uptakecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target gene, wherein the siNA construct comprises one ormore chemical modifications described herein that increases thebioavailability of the siNA construct, for example, by attachingpolymeric conjugates such as polyethyleneglycol or equivalent conjugatesthat improve the pharmacokinetics of the siNA construct, or by attachingconjugates that target specific tissue types or cell types in vivo.Non-limiting examples of such conjugates are described in Vargeese etal., U.S. Ser. No. 10/201,394 incorporated by reference herein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability comprising (a)introducing a conjugate into the structure of a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; polyamines, such as spermine or spermidine;and others.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or is recognized by cellular proteinsthat facilitate RNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. Such design or modifications are expected to enhance theactivity of siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In another embodiment, theterminal cap moiety comprises an inverted abasic, inverted deoxy abasic,inverted nucleotide moiety, a group shown in FIG. 22, an alkyl orcycloalkyl group, a heterocycle, or any other group that prevents RNAiactivity in which the second sequence serves as a guide sequence ortemplate for RNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In another embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.22, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of a siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 22(e.g. inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g. the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, a siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10” and “Stab 7/8” chemistries and variantsthereof wherein the 5′-end and 3′-end of the sense strand of the siNA donot comprise a hydroxyl group or phosphate group.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of a siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In another embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10” and “Stab 7/8” chemistries and variants thereof whereinthe 5′-end and 3′-end of the sense strand of the siNA do not comprise ahydroxyl group or phosphate group.

In one embodiment, the invention features a method for screening siNAmolecules against a target nucleic acid sequence comprising, (a)generating a plurality of unmodified siNA molecules, (b) assaying thesiNA molecules of step (a) under conditions suitable for isolating siNAmolecules that are active in mediating RNA interference against thetarget nucleic acid sequence, (c) introducing chemical modifications(e.g. chemical modifications as described herein or as otherwise knownin the art) into the active siNA molecules of (b), and (d) optionallyre-screening the chemically modified siNA molecules of (c) underconditions suitable for isolating chemically modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screening siNAmolecules against a target nucleic acid sequence comprising, (a)generating a plurality of chemically modified siNA molecules (e.g. siNAmolecules as described herein or as otherwise known in the art), and (b)assaying the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 2,000 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include a siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). Non limiting examples ofsiNA molecules of the invention are shown in FIGS. 18A-18F, 19A-19F, and20, and Table I herein. For example the siNA can be a double-strandedpolynucleotide molecule comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof. The siNA can be assembled from two separateoligonucleotides, where one strand is the sense strand and the other isthe antisense strand, wherein the antisense and sense strands areself-complementary (i.e. each strand comprises nucleotide sequence thatis complementary to nucleotide sequence in the other strand; such aswhere the antisense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 19 base pairs); the antisense strand comprises nucleotide sequencethat is complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof and the sense strand comprises nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. Alternatively, the siNA is assembled from a singleoligonucleotide, where the self-complementary sense and antisenseregions of the siNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s). The siNA can be a polynucleotide witha duplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The siNA can be a circular single-stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siNA molecule capable of mediating RNAi. The siNA can alsocomprise a single stranded polynucleotide having nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic intercations, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure to alter gene expression (see, for example,Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complimentary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 19 to about 22 nucleotides) and a loop region comprisingabout 4 to about 8 nucleotides, and a sense region having about 3 toabout 18 nucleotides that are complementary to the antisense region (seefor example FIG. 74). The asymmetric hairpin siNA molecule can alsocomprise a 5′-terminal phosphate group that can be chemically modified(for example as shown in FIG. 75). The loop portion of the asymmetrichairpin siNA molecule can comprise nucleotides, non-nucleotides, linkermolecules, or conjugate molecules as described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complimentarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 19 to about 22 nucleotides) and asense region having about 3 to about 18 nucleotides that arecomplementary to the antisense region (see for example FIG. 74).

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins orprotein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence.

By “gene”, or “target gene”, is meant, a nucleic acid that encodes anRNA, for example, nucleic acid sequences including, but not limited to,structural genes encoding a polypeptide. A gene or target gene can alsoencode a functional RNA (fRNA) or non-coding RNA (ncRNA), such as smalltemporal RNA (stRNA), micro RNA (miRNA), small nuclear RNA (snRNA),short interfering RNA (siRNA), small nucleolar RNA (snRNA), ribosomalRNA (rRNA), transfer RNA (tRNA) and precursor RNAs thereof. Suchnon-coding RNAs can serve as target nucleic acid molecules for siNAmediated RNA interference in modulating the activity of fRNA or ncRNAinvolved in functional or regulatory cellular processes. Abberant fRNAor ncRNA activity leading to disease can therefore be modulated by siNAmolecules of the invention. siNA molecules targeting fRNA and ncRNA canalso be used to manipulate or alter the genotype or phenotype of anorganism or cell, by intervening in cellular processes such as geneticimprinting, transcription, translation, or nucleic acid processing(e.g., transamination, methylation etc.). The target gene can be a genederived from a cell, an endogenous gene, a transgene, or exogenous genessuch as genes of a pathogen, for example a virus, which is present inthe cell after infection thereof. The cell containing the target genecan be derived from or contained in any organism, for example a plant,animal, protozoan, virus, bacterium, or fungus. Non-limiting examples ofplants include monocots, dicots, or gymnosperms. Non-limiting examplesof animals include vertebrates or invertebrates. Non-limiting examplesof fungi include molds or yeasts.

By “highly conserved sequence region” is meant, a nucleotide sequence ofone or more regions in a target gene does not vary significantly fromone generation to the other or from one biological system to the other.

By “cancer” is meant a group of diseases characterized by uncontrolledgrowth and spread of abnormal cells.

By “sense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of a siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence.

By “antisense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to a target nucleic acid sequence. In addition,the antisense region of a siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule.

By “target nucleic acid” is meant any nucleic acid sequence whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA, such as endogenous DNA or RNA, viral DNA or viral RNA, orother RNA encoded by a gene, virus, bacteria, fungus, mammal, or plant.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types. In reference to the nucleicmolecules of the present invention, the binding free energy for anucleic acid molecule with its complementary sequence is sufficient toallow the relevant function of the nucleic acid to proceed, e.g., RNAiactivity. Determination of binding free energies for nucleic acidmolecules is well known in the art (see, e.g., Turner et al., 1987, CSHSymp. Quant. Biol. LII pp. 123-133; Frier et al., 1986, Proc. Nat. Acad.Sci. USA 83:9373-9377; Turner et al., 1987, J. Am. Chem. Soc.109:3783-3785). A percent complementarity indicates the percentage ofcontiguous residues in a nucleic acid molecule that can form hydrogenbonds (e.g., Watson-Crick base pairing) with a second nucleic acidsequence (e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10nucleotides in the first oligonuelcotide being based paired to a secondnucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%,80%, 90%, and 100% complementary respectively). “Perfectlycomplementary” means that all the contiguous residues of a nucleic acidsequence will hydrogen bond with the same number of contiguous residuesin a second nucleic acid sequence.

The siNA molecules of the invention represent a novel therapeuticapproach to a broad spectrum of diseases and conditions, includingcancer or cancerous disease, infectious disease, cardiovascular disease,neurological disease, prion disease, inflammatory disease, autoimmunedisease, pulmonary disease, renal disease, liver disease, mitochondrialdisease, endocrine disease, reproduction related diseases andconditions, and any other indications that can respond to the level ofan expressed gene product in a cell or organism.

In one embodiment of the present invention, each sequence of a siNAmolecule of the invention is independently about 18 to about 24nucleotides in length, in specific embodiments about 18, 19, 20, 21, 22,23, or 24 nucleotides in length. In another embodiment, the siNAduplexes of the invention independently comprise about 17 to about 23base pairs (e.g., about 17, 18, 19, 20, 21, 22 or 23). In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., 38, 39, 40,41, 42, 43 or 44) nucleotides in length and comprising about 16 to about22 (e.g., about 16, 17, 18, 19, 20, 21 or 22) base pairs. Exemplary siNAmolecules of the invention are shown in Table I. and/or FIGS. 18A-18Fand 19A-19F.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through injection, infusion pump or stent, with or without theirincorporation in biopolymers. In particular embodiments, the nucleicacid molecules of the invention comprise sequences shown in Table Iand/or FIGS. 18A-18F and 19A-19F. Examples of such nucleic acidmolecules consist essentially of sequences defined in these tables andfigures. Furthermore, the chemically modified constructs described inTable IV can be applied to any siNA sequence of the invention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a 3-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter, that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate internucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an acyclic ribose sugar, for example where any of the ribosecarbons (C1, C2, C3, C4, or C5), are independently or in combinationabsent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to treatdiseases or conditions discussed herein (e.g., cancers and otherproliferative conditions, viral infection, inflammatory disease,autoimmunity, pulmonary disease, renal disease, ocular disease, etc.).For example, to treat a particular disease or condition, the siNAmolecules can be administered to a subject or can be administered toother appropriate cells evident to those skilled in the art,individually or in combination with one or more drugs under conditionssuitable for the treatment.

In one embodiment, the invention features a method for treating orpreventing a disease or condition in a subject, wherein the disease orcondition is related to angiogenesis or neovascularization, comprisingadministering to the subject a siNA molecule of the invention underconditions suitable for the treatment or prevention of the disease orcondition in the subject, alone or in conjunction with one or more othertherapeutic compounds. In another embodiment, the disease or conditioncomprises tumor angiogenesis and cancer, including but not limited tobreast cancer, lung cancer (including non-small cell lung carcinoma),prostate cancer, colorectal cancer, brain cancer, esophageal cancer,bladder cancer, pancreatic cancer, cervical cancer, head and neckcancer, skin cancers, nasopharyngeal carcinoma, liposarcoma, epithelialcarcinoma, renal cell carcinoma, gallbladder adeno carcinoma, parotidadenocarcinoma, ovarian cancer, melanoma, lymphoma, glioma, endometrialsarcoma, multidrug resistant cancers, diabetic retinopathy, maculardegeneration, age related macular degeneration, neovascular glaucoma,myopic degeneration, arthritis, psoriasis, endometriosis, femalereproduction, verruca vulgaris, angiofibroma of tuberous sclerosis,pot-wine stains, Sturge Weber syndrome, Kippel-Trenaunay-Weber syndrome,Osler-Weber-Rendu syndrome, renal disease such as Autosomal dominantpolycystic kidney disease (ADPKD), restenosis, arteriosclerosis, and anyother diseases or conditions that are related to gene expression or willrespond to RNA interference in a cell or tissue, alone or in combinationwith other therapies.

In one embodiment, the invention features a method for treating orpreventing an ocular disease or condition in a subject, wherein theocular disease or condition is related to angiogenesis orneovascularization, comprising administering to the subject a siNAmolecule of the invention under conditions suitable for the treatment orprevention of the disease or condition in the subject, alone or inconjunction with one or more other therapeutic compounds. In anotherembodiment, the ocular disease or condition comprises maculardegeneration, age related macular degeneration, diabetic retinopathy,neovascular glaucoma, myopic degeneration, trachoma, scarring of theeye, cataract, ocular inflammation and/or ocular infections.

In one embodiment, the invention features a method for treating orpreventing tumor angiogenesis in a subject, comprising administering tothe subject a siNA molecule of the invention under conditions suitablefor the treatment or prevention of tumor angiogenesis in the subject,alone or in conjunction with one or more other therapeutic compounds.

In one embodiment, the invention features a method for treating orpreventing viral infection or replication in a subject, comprisingadministering to the subject a siNA molecule of the invention underconditions suitable for the treatment or prevention of viral infectionor replication in the subject, alone or in conjunction with one or moreother therapeutic compounds.

In one embodiment, the invention features a method for treating orpreventing autoimmune disease in a subject, comprising administering tothe subject a siNA molecule of the invention under conditions suitablefor the treatment or prevention of autoimmune disease in the subject,alone or in conjunction with one or more other therapeutic compounds.

In one embodiment, the invention features a method for treating orpreventing inflammation in a subject, comprising administering to thesubject a siNA molecule of the invention under conditions suitable forthe treatment or prevention of inflammation in the subject, alone or inconjunction with one or more other therapeutic compounds.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to treat conditions or diseases discussedabove. For example, the described molecules could be used in combinationwith one or more known therapeutic agents to treat a disease orcondition. Non-limiting examples of other therapeutic agents that can bereadily combined with a siNA molecule of the invention are enzymaticnucleic acid molecules, allosteric nucleic acid molecules, antisense,decoy, or aptamer nucleic acid molecules, antibodies such as monoclonalantibodies, small molecules, and other organic and/or inorganiccompounds including metals, salts and ions.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form asiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows the results of a stability assay used to determine theserum stability of chemically modified siNA constructs compared to asiNA control consisting of all RNA with 3′-TT termini. T ½ values areshown for duplex stability.

FIG. 4 shows the results of an RNAi activity screen of severalphosphorothioate modified siNA constructs using a luciferase reportersystem.

FIG. 5 shows the results of an RNAi activity screen of severalphosphorothioate and universal base modified siNA constructs using aluciferase reporter system.

FIG. 6 shows the results of an RNAi activity screen of several2′-O-methyl modified siNA constructs using a luciferase reporter system.

FIG. 7 shows the results of an RNAi activity screen of several2′-O-methyl and 2′-deoxy-2′-fluoro modified siNA constructs using aluciferase reporter system.

FIG. 8 shows the results of an RNAi activity screen of aphosphorothioate modified siNA construct using a luciferase reportersystem.

FIG. 9 shows the results of an RNAi activity screen of an inverteddeoxyabasic modified siNA construct generated via tandem synthesis usinga luciferase reporter system.

FIG. 10 shows the results of an RNAi activity screen of chemicallymodified siNA constructs including 3′-glyceryl modified siNA constructscompared to an all RNA control siNA construct using a luciferasereporter system. These chemically modified siNAs were compared in theluciferase assay described herein at 1 nM and 10 nM concentration usingan all RNA siNA control (siGL2) having 3′-terminal dithymidine (TT) andits corresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I.

FIG. 11 shows the results of an RNAi activity screen of chemicallymodified siNA constructs. The screen compared various combinations ofsense strand chemical modifications and antisense strand chemicalmodifications. These chemically modified siNAs were compared in theluciferase assay described herein at 1 nM and 10 nM concentration usingan all RNA siNA control (siGL2) having 3′-terminal dithymidine (TT) andits corresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I.

FIG. 12 shows the results of an RNAi activity screen of chemicallymodified siNA constructs. The screen compared various combinations ofsense strand chemical modifications and antisense strand chemicalmodifications. These chemically modified siNAs were compared in theluciferase assay described herein at 1 nM and 10 nM concentration usingan all RNA siNA control (siGL2) having 3′-terminal dithymidine (TT) andits corresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I. In addition, the antisense strand alone (Sirna/RPI 30430) andan inverted control (Sirna/RPI 30227/30229, having matched chemistry toSirna/RPI (30063/30224) was compared to the siNA duplexes describedabove.

FIG. 13 shows the results of an RNAi activity screen of chemicallymodified siNA constructs. The screen compared various combinations ofsense strand chemical modifications and antisense strand chemicalmodifications. These chemically modified siNAs were compared in theluciferase assay described herein at 1 nM and 10 nM concentration usingan all RNA siNA control (siGL2) having 3′-terminal dithymidine (TT) andits corresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I. In addition, an inverted control (Sirna/RPI 30226/30229),having matched chemistry to Sirna/RPI (30222/30224) was compared to thesiNA duplexes described above.

FIG. 14 shows the results of an RNAi activity screen of chemicallymodified siNA constructs including various 3′-terminal modified siNAconstructs compared to an all RNA control siNA construct using aluciferase reporter system. These chemically modified siNAs werecompared in the luciferase assay described herein at 1 nM and 10 nMconcentration using an all RNA siNA control (siGL2) having 3′-terminaldithymidine (TT) and its corresponding inverted control (Inv siGL2). Thebackground level of luciferase expression in the HeLa cells isdesignated by the “cells” column. Sense and antisense strands ofchemically modified siNA constructs are shown by Sirna/RPI number (sensestrand/antisense strand). Sequences corresponding to these Sirna/RPInumbers are shown in Table I.

FIG. 15 shows the results of an RNAi activity screen of chemicallymodified siNA constructs. The screen compared various combinations ofsense strand chemistries compared to a fixed antisense strand chemistry.These chemically modified siNAs were compared in the luciferase assaydescribed herein at 1 nM and 10 nM concentration using an all RNA siNAcontrol (siGL2) having 3′-terminal dithymidine (TT) and itscorresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I.

FIG. 16 shows the results of a siNA titration study using a luciferasereporter system, wherein the RNAi activity of a phosphorothioatemodified siNA construct is compared to that of a siNA constructconsisting of all ribonucleotides except for two terminal thymidineresidues.

FIG. 17 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIGS. 18A-18F shows non-limiting examples of chemically-modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs.

FIG. 18A: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety and wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all nucleotidespresent are ribonucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”connects the (N N) nucleotides in the antisense strand.

FIG. 18B: The sense strand comprises 21 nucleotides wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. The antisense strand comprises21 nucleotides, optionally having a 3′-terminal glyceryl moiety andwherein the two terminal 3′-nucleotides are optionally complementary tothe target RNA sequence, and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides and all purinenucleotides that may be present are 2′-O-methyl modified nucleotidesexcept for (N N) nucleotides, which can comprise ribonucleotides,deoxynucleotides, universal bases, or other chemical modificationsdescribed herein. A modified internucleotide linkage, such as aphosphorothioate, phosphorodithioate or other modified internucleotidelinkage as described herein, shown as “s” connects the (N N) nucleotidesin the sense and antisense strand. FIG. 18C: The sense strand comprises21 nucleotides having 5′- and 3′-terminal cap moieties wherein the twoterminal 3′-nucleotides are optionally base paired and wherein allpyrimidine nucleotides that may be present are 2′-O-methyl or2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety and wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all pyrimidinenucleotides that may be present are 2′-deoxy-2′-fluoro modifiednucleotides except for (N N) nucleotides, which can compriseribonucleotides, deoxynucleotides, universal bases, or other chemicalmodifications described herein. A modified internucleotide linkage, suchas a phosphorothioate, phosphorodithioate or other modifiedinternucleotide linkage as described herein, shown as “s” connects the(N N) nucleotides in the antisense strand. FIG. 18D: The sense strandcomprises 21 nucleotides having 5′- and 3′-terminal cap moieties whereinthe two terminal 3′-nucleotides are optionally base paired and whereinall pyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides except for (N N) nucleotides, which can compriseribonucleotides, deoxynucleotides, universal bases, or other chemicalmodifications described herein and wherein and all purine nucleotidesthat may be present are 2′-deoxy nucleotides. The antisense strandcomprises 21 nucleotides, optionally having a 3′-terminal glycerylmoiety and wherein the two terminal 3′-nucleotides are optionallycomplementary to the target RNA sequence, and wherein all pyrimidinenucleotides that may be present are 2′-deoxy-2′-fluoro modifiednucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”connects the (N N) nucleotides in the antisense strand.

FIG. 18E: The sense strand comprises 21 nucleotides having 5′- and3′-terminal cap moieties wherein the two terminal 3′-nucleotides areoptionally base paired and wherein all pyrimidine nucleotides that maybe present are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”connects the (N N) nucleotides in the antisense strand. FIG. 18F: Thesense strand comprises 21 nucleotides having 5′- and 3′-terminal capmoieties wherein the two terminal 3′-nucleotides are optionally basepaired and wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein and wherein and allpurine nucleotides that may be present are 2′-deoxy nucleotides. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-deoxy nucleotides except for (N N) nucleotides, which can compriseribonucleotides, deoxynucleotides, universal bases, or other chemicalmodifications described herein. A modified internucleotide linkage, suchas a phosphorothioate, phosphorodithioate or other modifiedinternucleotide linkage as described herein, shown as “s” connects the(N N) nucleotides in the antisense strand. The antisense strand ofconstructs A-F comprise sequence complementary to any target nucleicacid sequence of the invention. Furthermore, when a glyceryl moiety (L)is present at the 3′-end of the antisense strand for any construct shownin FIG. 4 A-F, the modified internucleotide linkage is optional.

FIGS. 19A-19F shows non-limiting examples of specific chemicallymodified siNA sequences of the invention. FIGS. 19A-19F apply thechemical modifications described in FIGS. 18A-18F to a representativesiNA sequence targeting the hepatitis C virus (HCV).

FIG. 20 shows non-limiting examples of different siNA constructs of theinvention. The examples shown (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example comprising about 1,2, 3, or 4 nucleotides in length when present, preferably about 2nucleotides. Such overhangs can be present or absent (i.e., blunt ends).Such blunt ends can be present on one end or both ends of the siNAmolecule, for example where all nucleotides present in a siNA duplex arebase paired. Constructs 1 and 2 can be used independently for RNAiactivity. Construct 2 can comprise a polynucleotide or non-nucleotidelinker, which can optionally be designed as a biodegradable linker. Inone embodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

FIGS. 21A-21D are a diagrammatic representation of a method used todetermine target sites for siNA mediated RNAi within a particular targetnucleic acid sequence, such as messenger RNA. (FIG. 21A) A pool of siNAoligonucleotides are synthesized wherein the antisense region of thesiNA constructs has complementarity to target sites across the targetnucleic acid sequence, and wherein the sense region comprises sequencecomplementary to the antisense region of the siNA. (FIG. 21B) Thesequences are transfected into cells. (FIG. 21C) Cells are selectedbased on phenotypic change that is associated with modulation of thetarget nucleic acid sequence. (FIG. 21D) The siNA is isolated from theselected cells and is sequenced to identify efficacious target siteswithin the target nucleic acid sequence.

FIG. 22 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 23 shows a non-limiting example of siNA mediated inhibition ofVEGF-induced angiogenesis using the rat corneal model of angiogenesis.siNA targeting site 2340 of VEGFR1 RNA (shown as Sirna/RPI No.29695/29699) were compared to inverted controls (shown as Sirna/RPI No.29983/29984) at three different concentrations and compared to a VEGFcontrol in which no siNA was administered.

FIG. 24 is a non-limiting example of a HBsAg screen of stabilized siNAconstructs (“stab 4/5”, see Table IV) targeting HBV pregenomic RNA inHepG2 cells at 25 nM compared to untreated and matched chemistryinverted sequence controls. The siNA sense and antisense strands areshown by Sirna/RPI number (sense/antisense).

FIG. 25 is a non-limiting example of a dose response HBsAg screen ofstabilized siNA constructs (“stab 4/5”, see Table IV) targeting sites262 and 1580 of the HBV pregenomic RNA in HepG2 cells at 0.5, 5, 10 and25 nM compared to untreated and matched chemistry inverted sequencecontrols. The siNA sense and antisense strands are shown by Sirna/RPInumber (sense/antisense).

FIG. 26 shows a dose response comparison of two different stabilizationchemistries (“stab 7/8” and “stab 7/11”, see Table IV) targeting site1580 of the HBV pregenomic RNA in HepG2 cells at 5, 10, 25, 50 and 100nM compared to untreated and matched chemistry inverted sequencecontrols. The siNA sense and antisense strands are shown by Sirna/RPInumber (sense/antisense).

FIG. 27 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistance while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g. introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 28 shows representative data of a chemically modified siNAconstruct (Stab 4/5, Table IV) targeting HBV site 1580 RNA compared toan unstabilized siRNA construct in a dose response time course HBsAgassay. The constructs were compared at different concentrations (5 nM,10 nM, 25 nM, 50 nM, and 100 nM) over the course of nine days. Activitybased on HBsAg levels was determined at day 3, day 6, and day 9.

FIG. 29 shows representative data of a chemically modified siNAconstruct (Stab 7/8, Table IV) targeting HBV site 1580 RNA compared toan unstabilized siRNA construct in a dose response time course HBsAgassay. The constructs were compared at different concentrations (5 nM,10 nM, 25 nM, 50 nM, and 100 nM) over the course of nine days. SiNAactivity based on HBsAg levels was determined at day 3, day 6, and day9.

FIG. 30 shows representative data of a chemically modified siNAconstruct (Stab 7/11, Table IV) targeting HBV site 1580 RNA compared toan unstabilized siRNA construct in a dose response time course HBsAgassay. The constructs were compared at different concentrations (5 nM,10 nM, 25 nM, 50 nM, and 100 nM) over the course of nine days. SiNAactivity based on HBsAg levels was determined at day 3, day 6, and day9.

FIG. 31 shows representative data of a chemically modified siNAconstruct (Stab 9/10, Table IV) targeting HBV site 1580 RNA compared toan unstabilized siRNA construct in a dose response time course HBsAgassay. The constructs were compared at different concentrations (5 nM,10 nM, 25 nM, 50 nM, and 100 nM) over the course of nine days. SiNAactivity based on HBsAg levels was determined at day 3, day 6, and day9.

FIG. 32 shows non-limiting examples of inhibition of viral replicationof a HCV/poliovirus chimera by siNA constructs targeted to HCV chimera(29579/29586; 29578/29585) compared to control (29593/29600).

FIG. 33 shows a non-limiting example of a dose response studydemonstrating the inhibition of viral replication of a HCV/polioviruschimera by siNA construct (29579/29586) at various concentrations (1 nM,5 nM, 10 nM, and 25 nM) compared to control (29593/29600).

FIG. 34 shows a non-limiting example demonstrating the inhibition ofviral replication of a HCV/poliovirus chimera by a chemically modifiedsiRNA construct (30051/30053) compared to control construct(30052/30054).

FIG. 35 shows a non-limiting example demonstrating the inhibition ofviral replication of a HCV/poliovirus chimera by a chemically modifiedsiRNA construct (30055/30057) compared to control construct(30056/30058).

FIG. 36 shows a non-limiting example of several chemically modifiedsiRNA constructs targeting viral replication of an HCV/polioviruschimera at 10 nM treatment in comparison to a lipid control and aninverse siNA control construct 29593/29600.

FIG. 37 shows a non-limiting example of several chemically modifiedsiRNA constructs targeting viral replication of a HCV/poliovirus chimeraat 25 nM treatment in comparison to a lipid control and an inverse siNAcontrol construct 29593/29600.

FIG. 38 shows a non-limiting example of several chemically modifiedsiRNA constructs targeting viral replication of a Huh7 HCV repliconsystem at 25 nM treatment in comparison to untreated cells (“cells”),cells transfected with lipofectamine (“LFA2K”) and inverse siNA controlconstructs.

FIG. 39 shows a non-limiting example of a dose response study usingchemically modified siNA molecules (Stab 4/5, see Table IV) targetingHCV RNA sites 291, 300, and 303 in a Huh7 HCV replicon system at 5, 10,25, and 100 nM treatment comparison to untreated cells (“cells”), cellstransfected with lipofectamine (“LFA”) and inverse siNA controlconstructs.

FIG. 40 shows a non-limiting example of several chemically modified siNAconstructs (Stab 7/8, see Table IV) targeting viral replication in aHuh7 HCV replicon system at 25 nM treatment in comparison to untreatedcells (“cells”), cells transfected with lipofectamine (“Lipid”) andinverse siNA control constructs.

FIG. 41 shows a non-limiting example of a dose response study usingchemically modified siNA molecules (Stab 7/8, see Table IV) targetingHCV site 327 in a Huh7 HCV replicon system at 5, 10, 25, 50, and 100 nMtreatment in comparison to inverse siNA control constructs.

FIG. 42 shows a synthetic scheme for post-synthetic modification of anucleic acid molecule to produce a folate conjugate.

FIG. 43 shows a synthetic scheme for generating an oligonucleotide ornucleic acid-folate conjugate.

FIG. 44 shows an alternative synthetic scheme for generating anoligonucleotide or nucleic acid-folate conjugate.

FIG. 45 shows an alternative synthetic scheme for post-syntheticmodification of a nucleic acid molecule to produce a folate conjugate.

FIG. 46 shows a non-limiting example of a synthetic scheme for thesynthesis of a N-acetyl-D-galactosamine-2′-aminouridine phosphoramiditeconjugate of the invention.

FIG. 47 shows a non-limiting example of a synthetic scheme for thesynthesis of a N-acetyl-D-galactosamine-D-threoninol phosphoramiditeconjugate of the invention.

FIG. 48 shows a non-limiting example of a N-acetyl-D-galactosamine siNAnucleic acid conjugate of the invention. W shown in the example refersto a biodegradable linker, for example a nucleic acid dimer, trimer, ortetramer comprising ribonucleotides and/or deoxyribonucleotides. ThesiNA can be conjugated at the 3′, 5′ or both 3′ and 5′ ends of the sensestrand of a double stranded siNA and/or the 3′-end of the antisensestrand of the siNA. A single stranded siNA molecule can be conjugated atthe 3′-end of the siNA.

FIG. 49 shows a non-limiting example of a synthetic scheme for thesynthesis of a dodecanoic acid derived conjugate linker of theinvention.

FIG. 50 shows a non-limiting example of a synthetic scheme for thesynthesis of an oxime linked nucleic acid/peptide conjugate of theinvention. FIG. 50 discloses “SGACRGDCLGA” as SEQ ID NO: 684 and“GACRGDCLGA” as SEQ ID NO: 685.

FIG. 51 shows non-limiting examples of phospholipid derived siNAconjugates of the invention. CL shown in the examples refers to abiodegradable linker, for example a nucleic acid dimer, trimer, ortetramer comprising ribonucleotides and/or deoxyribonucleotides. ThesiNA can be conjugated at the 3′, 5′ or both 3′ and 5′ ends of the sensestrand of a double stranded siNA and/or the 3′-end of the antisensestrand of the siNA. A single stranded siNA molecule can be conjugated atthe 3′-end of the siNA.

FIG. 52 shows a non-limiting example of a synthetic scheme for preparinga phospholipid derived siNA conjugates of the invention.

FIG. 53 shows a non-limiting example of a synthetic scheme for preparinga poly-N-acetyl-D-galactosamine nucleic acid conjugate of the invention.

FIG. 54 shows a non-limiting example of the synthesis of siNAcholesterol conjugates of the invention using a phosphoramiditeapproach.

FIG. 55 shows a non-limiting example of the synthesis of siNA PEGconjugates of the invention using NHS ester coupling.

FIG. 56 shows a non-limiting example of the synthesis of siNAcholesterol conjugates of the invention using NHS ester coupling.

FIG. 57 shows a non-limiting example of various siNA cholesterolconjugates of the invention.

FIG. 58 shows a non-limiting example of various siNA cholesterolconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

FIG. 59 shows a non-limiting example of various siNA cholesterolconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

FIG. 60 shows a non-limiting example of various siNA cholesterolconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule.

FIG. 61 shows a non-limiting example of various siNA phospholipidconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

FIG. 62 shows a non-limiting example of various siNA phospholipidconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule.

FIG. 63 shows a non-limiting example of various siNA galactosamineconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule.

FIG. 64 shows a non-limiting example of various siNA galactosamineconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule.

FIG. 65 shows a non-limiting example of various generalized siNAconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a doublestranded siNA molecule. CONJ in the figure refers to any biologicallyactive compound or any other conjugate compound as described herein andin the Formulae herein.

FIG. 66 shows a non-limiting example of various generalized siNAconjugates of the invention in which various linker chemistries and/orcleavable linkers can be utilized at different positions of a singlestranded siNA molecule. CONJ in the figure refers to any biologicallyactive compound or any other conjugate compound as described herein andin the Formulae herein.

FIG. 67 shows a non-limiting example of the pharmacokinetic distributionof intact siNA in liver after administration of conjugated orunconjugated siNA molecules in mice.

FIG. 68 shows a non-limiting example of the activity of conjugated siNAconstructs compared to matched chemistry unconjugated siNA constructs inan HBV cell culture system without the use of transfection lipid. Asshown in the Figure, siNA conjugates provide efficacy in cell culturewithout the need for transfection reagent.

FIG. 69 shows a non-limiting example of a scheme for the synthesis of amono-galactosamine phosphoramidite of the invention that can be used togenerate galactosamine conjugated nucleic acid molecules.

FIG. 70 shows a non-limiting example of a scheme for the synthesis of atri-galactosamine phosphoramidite of the invention that can be used togenerate tri-galactosamine conjugated nucleic acid molecules.

FIG. 71 shows a non-limiting example of a scheme for the synthesis ofanother tri-galactosamine phosphoramidite of the invention that can beused to generate tri-galactosamine conjugated nucleic acid molecules.

FIG. 72 shows a non-limiting example of an alternate scheme for thesynthesis of a tri-galactosamine phosphoramidite of the invention thatcan be used to generate tri-galactosamine conjugated nucleic acidmolecules.

FIG. 73 shows a non-limiting example of a scheme for the synthesis of acholesterol NHS ester of the invention that can be used to generatecholesterol conjugated nucleic acid molecules.

FIG. 74 shows non-limiting exampled of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 75 shows non-limiting examples of a chemically modified terminalphosphate groups of the invention.

FIG. 76 shows a non-limiting example of inhibition of VEGF inducedneovascularization in the rat corneal model. VEGFr1 site 349 active siNAhaving “Stab 9/10” chemistry (Sirna #31270/31273) was tested forinhibition of VEGF-induced angiogenesis at three differentconcentrations (2.0 ug, 1.0 ug, and 0.1 μg dose response) as compared toa matched chemistry inverted control siNA construct (Sirna #31276/31279)at each concentration and a VEGF control in which no siNA wasadministered. As shown in the figure, the active siNA construct having“Stab 9/10” chemistry (Sima #31270/31273) is highly effective ininhibiting VEGF-induced angiogenesis in the rat corneal model comparedto the matched chemistry inverted control siNA at concentrations from0.1 μg to 2.0 ug.

FIGS. 77A-77F show activity of modified siNA constructs having stab 4/5(Sirna 30355/30366), stab 7/8 (Sirna 30612/30620), and stab 7/11 (Sirna30612/31175) chemistries and an all ribo siNA construct (Sirna30287/30298) in the reduction of HBsAg levels compared to matchedinverted controls at FIG. 77A. 3 days, FIG. 77B. 9 days, and FIG. 77C.21 days post transfection. Also shown is the corresponding percentinhibition as function of time at siNA concentrations of FIG. 77D. 100nM, FIG. 77E. 50 nM, and FIG. 77F. 25 nM.

FIG. 78 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 79 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 80 shows a non-limiting example of reduction of serum HBV DNA inmice treated with hydrodynamically administered chemically modified siNA(Stab 7/8 and Stab 9/10) targeting HBV RNA compared to matched chemistryinverted controls and a saline control.

FIG. 81 shows a non-limiting example of reduction of serum HBV S antigen(HBsAg) in mice treated with hydrodynamically administered chemicallymodified siNA (Stab 7/8 and Stab 9/10) targeting HBV RNA compared tomatched chemistry inverted controls and a saline control.

FIG. 82 shows a non-limiting example of reduction of serum HBV RNA inmice treated with hydrodynamically administered chemically modified siNA(Stab 7/8 and Stab 9/10) targeting HBV RNA compared to matched chemistryinverted constrols and a saline control.

FIG. 83 shows a non-limiting example of reduction of serum HBV DNA inmice treated with hydrodynamically administered chemically modified siNA(Stab 7/8 and Stab 9/10) targeting HBV RNA at 5 days and 7 days postadministration.

FIG. 84 shows a non-limiting example of an assay for dose dependentreduction of Luciferase expression utilizing Stab 7/8 chemicallymodified siNA constructs targeting luciferase RNA sites 80, 237, and1478 that were selected from a screen using all Stab 7/8 chemicallymodified siNA constructs.

FIG. 85 shows a non-limiting example of an assay for dose dependentreduction of Luciferase expression utilizing Stab 7/8 chemicallymodified siNA constructs targeting luciferase RNA sites 1544 and 1607that were selected from a screen using all Stab 7/8 chemically modifiedsiNA constructs.

FIG. 86 shows a non-limiting example of an assay screen of Stab 7/8 siNAconstructs targeting various sites of HCV RNA in a replicon systemcompared to untreated, lipid, and an inverted control. As shown in thefigure, several Stab 7/8 constructs were identified with potent anti-HCVactivity as shown by reduction in HCV RNA levels.

FIG. 87 shows a non-limiting example of an assay screen of Stab 7/8 siNAconstructs targeting various sites of HBV RNA in HEpG2 cells compared tountreated cells and an inverted control. As shown in the figure, severalStab 7/8 constructs were identified with potent anti-HBV activity asshown by reduction in HBV S antigen levels.

FIG. 88 shows a non-limiting example of an assay screen of variouscombinations of chemically modified siNA constructs (e.g., Stab 7/8,7/10, 7/11, 9/8, and 9/10) targeting site 1580 of HBV RNA in HEpG2 cellscompared to untreated cells and an matched chemistry inverted controls.As shown in the figure, the combination chemistries tested demonstratedpotent anti-HBV activity as shown by reduction in HBV S antigen levels.

FIG. 89 shows a non-limiting example of an assay screen of variouscombinations of chemically modified siNA constructs (e.g., Stab 7/8,9/10, 6/10, 16/8, 16/10, 18/8, and 18/10) targeting site 1580 of HBV RNAin HEpG2 cells compared to untreated cells and an matched chemistryinverted controls. As shown in the figure, the combination chemistriestested demonstrated potent anti-HBV activity as shown by reduction inHBV S antigen levels.

FIG. 90 shows a non-limiting example of an assay screen of variouscombinations of chemically modified siNA constructs (e.g., Stab 4/8,4/10, 7/5, 7/10, 9/5, 9/8, and 9/11) targeting site 1580 of HBV RNA inHEpG2 cells compared to untreated cells and an matched chemistryinverted controls. As shown in the figure, the combination chemistriestested demonstrated potent anti-HBV activity as shown by reduction inHBV S antigen levels.

DETAILED DESCRIPTION OF THE INVENTION

Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically-modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitedto siRNA only and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or a siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806). The corresponding process in plants iscommonly referred to as post-transcriptional gene silencing or RNAsilencing and is also referred to as quelling in fungi. The process ofpost-transcriptional gene silencing is thought to be anevolutionarily-conserved cellular defense mechanism used to prevent theexpression of foreign genes which is commonly shared by diverse floraand phyla (Fire et al., 1999, Trends Genet., 15, 358). Such protectionfrom foreign gene expression may have evolved in response to theproduction of double-stranded RNAs (dsRNAs) derived from viral infectionor the random integration of transposon elements into a host genome viaa cellular response that specifically destroys homologoussingle-stranded RNA or viral genomic RNA. The presence of dsRNA in cellstriggers the RNAi response though a mechanism that has yet to be fullycharacterized. This mechanism appears to be different from theinterferon response that results from dsRNA-mediated activation ofprotein kinase PKR and 2′, 5′-oligoadenylate synthetase resulting innon-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing a siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of a siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table V outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM 12, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick &Jackson Synthesis Grade acetonitrile is used directly from the reagentbottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made upfrom the solid obtained from American International Chemical, Inc.Alternately, for the introduction of phosphorothioate linkages, Beaucagereagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile)is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table V outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mM12, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & JacksonSynthesis Grade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in acetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10minutes. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μL TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′, 4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample, proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

In one embodiment, the invention features a compound having Formula 1:

wherein each R₁, R₃, R₄, R₅, R₆, R₇ and R₈ is independently hydrogen,alkyl substituted alkyl, aryl, substituted aryl, or a protecting group,each “n” is independently an integer from 0 to about 200, R₁₂ is astraight or branched chain alkyl, substituted alkyl, aryl, orsubstituted aryl, and R₂ is a siNA molecule or a portion thereof.

In one embodiment, the invention features a compound having Formula 2:

wherein each R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₁₂ is a straightor branched chain alkyl, substituted alkyl, aryl, or substituted aryl,and R₂ is a siNA molecule or a portion thereof.

In one embodiment, the invention features a compound having Formula 3:

wherein each R₁, R₃, R₄, R₅ R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₁₂ is a straightor branched chain alkyl, substituted alkyl, aryl, or substituted aryl,and R₂ is a siNA molecule or a portion thereof.

In one embodiment, the invention features a compound having Formula 4:

wherein each R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₂ is a siNAmolecule or a portion thereof, and R₁₃ is an amino acid side chain.

In one embodiment, the invention features a compound having Formula 5:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆, R₇ and R₈ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl,or substituted aryl, and each R₉ and R₁₀ is independently a nitrogencontaining group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

In one embodiment, the invention features a compound having Formula 6:

wherein each R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, R₂ isa siNA molecule or a portion thereof, each “n” is independently aninteger from 0 to about 200, and L is a degradable linker.

In one embodiment, the invention features a compound having Formula 7:

wherein each R₁, R₃, R₄, R₅, R₆ and R₇ is independently hydrogen, alkyl,substituted alkyl, aryl, substituted aryl, or a protecting group, each“n” is independently an integer from 0 to about 200, R₁₂ is a straightor branched chain alkyl, substituted alkyl, aryl, or substituted aryl,and R₂ is a siNA molecule or a portion thereof.

In one embodiment, the invention features a compound having Formula 8:

wherein each R₁ and R₄ is independently a protecting group or hydrogen,each R₃, R₅, R₆ and R₇ is independently hydrogen, alkyl or nitrogenprotecting group, each “n” is independently an integer from 0 to about200, R₁₂ is a straight or branched chain alkyl, substituted alkyl, aryl,or substituted aryl, and each R₉ and R₁₀ is independently a nitrogencontaining group, cyanoalkoxy, alkoxy, aryloxy, or alkyl group.

In one embodiment, R₁₃ of a compound of the invention comprises analkylamino or an alkoxy group, for example, —CH₂O— or —CH(CH₂)CH₂O—.

In another embodiment, R₁₂ of a compound of the invention is analkylhyrdroxyl, for example, —(CH2)_(n)OH, where n comprises an integerfrom about 1 to about 10.

In another embodiment, L of Formula 6 of the invention comprises serine,threonine, or a photolabile linkage.

In one embodiment, R₉ of a compound of the invention comprises aphosphorus protecting group, for example —OCH₂CH₂CN (oxyethylcyano).

In one embodiment, R₁₀ of a compound of the invention comprises anitrogen containing group, for example, —N(R₁₄) wherein R₁₄ is astraight or branched chain alkyl having from about 1 to about 10carbons.

In another embodiment, R₁₀ of a compound of the invention comprises aheterocycloalkyl or heterocycloalkenyl ring containing from about 4 toabout 7 atoms, and having from about 1 to about 3 heteroatoms comprisingoxygen, nitrogen, or sulfur.

In another embodiment, R₁ of a compound of the invention comprises anacid labile protecting group, such as a trityl or substituted tritylgroup, for example, a dimethoxytrityl or mono-methoxytrityl group.

In another embodiment, R₄ of a compound of the invention comprises atert-butyl, Fm (fluorenyl-methoxy), or allyl group.

In one embodiment, R₆ of a compound of the invention comprises a TFA(trifluoracetyl) group.

In another embodiment, R₃, R₅ R₇ and R₈ of a compound of the inventionare independently hydrogen.

In one embodiment, R₇ of a compound of the invention is independentlyisobutyryl, dimethylformamide, or hydrogen.

In another embodiment, R₁₂ of a compound of the invention comprises amethyl group or ethyl group.

In one embodiment, the invention features a compound having Formula 27:

wherein “n” is an integer from about 0 to about 20, R₄ is H or acationic salt, X is a siNA molecule or a portion thereof, and R₂₄ is asulfur containing leaving group, for example a group comprising:

In one embodiment, the invention features a compound having Formula 39:

wherein “n” is an integer from about 0 to about 20, X is a siNA moleculeor a portion thereof, and P is a phosphorus containing group.

In another embodiment, a thiol containing linker of the invention is acompound having Formula 41:

wherein “n” is an integer from about 0 to about 20, P is a phosphoruscontaining group, for example a phosphine, phosphite, or phosphate, andR24 is any alkyl, substituted alkyl, alkoxy, aryl, substituted aryl,alkenyl, substituted alkenyl, alkynyl, or substituted alkynyl group withor without additional protecting groups.

In one embodiment, the invention features a compound having Formula 43:

wherein X comprises a siNA molecule or portion thereof; W comprises adegradable nucleic acid linker; Y comprises a linker molecule or aminoacid that can be present or absent; Z comprises H, OH, O-alkyl, SH,S-alkyl, alkyl, substituted alkyl, aryl, substituted aryl, amino,substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, amino acid, peptide, protein, lipid, phospholipid, orlabel; n is an integer from about 1 to about 100; and N′ is an integerfrom about 1 to about 20. In another embodiment, W is selected from thegroup consisting of amide, phosphate, phosphate ester, phosphoramidate,or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula44:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent; n isan integer from about 1 to about 50, and PEG represents a compoundhaving Formula 45:

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, or label; and n is an integer from about 1 to about100. In another embodiment, W is selected from the group consisting ofamide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

In another embodiment, the invention features a compound having Formula46:

wherein X comprises a siNA molecule or portion thereof; each Windependently comprises linker molecule or chemical linkage that can bepresent or absent, Y comprises a linker molecule or chemical linkagethat can be present or absent; and PEG represents a compound havingFormula 45:

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, or label; and n is an integer from about 1 to about100. In another embodiment, W is selected from the group consisting ofamide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

In one embodiment, the invention features a compound having Formula 47:

wherein X comprises a siNA molecule or portion thereof; each Windependently comprises a linker molecule or chemical linkage that canbe the same or different and can be present or absent, Y comprises alinker molecule that can be present or absent; each Q independentlycomprises a hydrophobic group or phospholipid; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and n is aninteger from about 1 to about 10. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula48:

wherein X comprises a siNA molecule or portion thereof; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and B represents a lipophilic group, for example asaturated or unsaturated linear, branched, or cyclic alkyl group,cholesterol, or a derivative thereof. In another embodiment, W isselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula49:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule that can be present or absent; each R1, R2,R3, and R4 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and Brepresents a lipophilic group, for example a saturated or unsaturatedlinear, branched, or cyclic alkyl group, cholesterol, or a derivativethereof. In another embodiment, W is selected from the group consistingof amide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

In another embodiment, the invention features a compound having Formula50:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule or chemical linkage that can be present orabsent; and each Q independently comprises a hydrophobic group orphospholipid. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 51:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent; Ycomprises a linker molecule or amino acid that can be present or absent;Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substituted alkyl, aryl,substituted aryl, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, or label; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, and n is aninteger from about 1 to about 20. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula52:

wherein X comprises a siNA molecule or portion thereof; Y comprises alinker molecule or chemical linkage that can be present or absent; eachR1, R2, R3, R4, and R5 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N; Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl,substituted alkyl, aryl, substituted aryl, amino, substituted amino,nucleotide, nucleoside, nucleic acid, oligonucleotide, amino acid,peptide, protein, lipid, phospholipid, or label; SG comprises a sugar,for example galactose, galactosamine, N-acetyl-galactosamine, glucose,mannose, fructose, or fucose and the respective D or L, alpha or betaisomers, n is an integer from about 1 to about 20; and N′ is an integerfrom about 1 to about 20. In another embodiment, X comprises a siNAmolecule or a portion thereof. In another embodiment, Y is selected fromthe group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula53:

wherein B comprises H, a nucleoside base, or a non-nucleosidic base withor without protecting groups; each R1 independently comprises O, N, S,alkyl, or substituted N; each R2 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; each R3 independently comprises N or O—N,each R4 independently comprises O, CH2, S, sulfone, or sulfoxy; Xcomprises H, a removable protecting group, a siNA molecule or a portionthereof; W comprises a linker molecule or chemical linkage that can bepresent or absent; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, each n isindependently an integer from about 1 to about 50; and N′ is an integerfrom about 1 to about 10. In another embodiment, W is selected from thegroup consisting of amide, phosphate, phosphate ester, phosphoramidate,or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula54:

wherein B comprises H, a nucleoside base, or a non-nucleosidic base withor without protecting groups; each R1 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; X comprises H, a removable protectinggroup, a siNA molecule or a portion thereof; W comprises a linkermolecule or chemical linkage that can be present or absent; and SGcomprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers. In another embodiment, W isselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 55:

wherein each R1 independently comprises O, N, S, alkyl, or substitutedN; each R2 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylhalo, S, N, substituted N, or a phosphorus containing group; eachR3 independently comprises H, OH, alkyl, substituted alkyl, or halo; Xcomprises H, a removable protecting group, a siNA molecule or a portionthereof; W comprises a linker molecule or chemical linkage that can bepresent or absent; SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine, glucose, mannose, fructose, orfucose and the respective D or L, alpha or beta isomers, each n isindependently an integer from about 1 to about 50; and N′ is an integerfrom about 1 to about 100. In another embodiment, W is selected from thegroup consisting of amide, phosphate, phosphate ester, phosphoramidate,or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula56:

wherein R1 comprises H, alkyl, alkylhalo, N, substituted N, or aphosphorus containing group; R2 comprises H, O, OH, alkyl, alkylhalo,halo, S, N, substituted N, or a phosphorus containing group; X comprisesH, a removable protecting group, a siNA molecule or a portion thereof; Wcomprises a linker molecule or chemical linkage that can be present orabsent; SG comprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine, glucose, mannose, fructose, or fucose and therespective D or L, alpha or beta isomers, and each n is independently aninteger from about 0 to about 20. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula57:

-   -   wherein R1 can include the groups:

-   -   and wherein R2 can include the groups:

and wherein Tr is a removable protecting group, for example a trityl,monomethoxytrityl, or dimethoxytrityl; SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers,and n is an integer from about 1 to about 20.

In one embodiment, compounds having Formula 52, 53, 54, 55, 56, and 57are featured wherein each nitrogen adjacent to a carbonyl canindependently be substituted for a carbonyl adjacent to a nitrogen oreach carbonyl adjacent to a nitrogen can be substituted for a nitrogenadjacent to a carbonyl.

In another embodiment, the invention features a compound having Formula58:X—W—[Y—(V)_(n)]_(N′)  58

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent; Ycomprises a linker molecule or amino acid that can be present or absent;V comprises a signal protein or peptide, for example Human serum albuminprotein, Antennapedia peptide, Kaposi fibroblast growth factor peptide,Caiman crocodylus Ig(5) light chain peptide, HIV envelope glycoproteingp41 peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; each n is independentlyan integer from about 1 to about 50; and N′ is an integer from about 1to about 100. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula59:

wherein each R1 independently comprises O, S, N, substituted N, or aphosphorus containing group; each R2 independently comprises O, S, or N;X comprises H, amino, substituted amino, nucleotide, nucleoside, nucleicacid, oligonucleotide, or other biologically active molecule; n is aninteger from about 1 to about 50, Q comprises H or a removableprotecting group which can be optionally absent, each W independentlycomprises a linker molecule or chemical linkage that can be present orabsent, and V comprises a signal protein or peptide, for example Humanserum albumin protein, Antennapedia peptide, Kaposi fibroblast growthfactor peptide, Caiman crocodylus Ig(5) light chain peptide, HIVenvelope glycoprotein gp41 peptide, HIV-1 Tat peptide, Influenzahemagglutinin envelope glycoprotein peptide, or transportan A peptide,or a compound having Formula 45

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, a removableprotecting group, a siNA molecule or a portion thereof; and n is aninteger from about 1 to about 100. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula60:

-   -   wherein R1 can include the groups:

-   -   and wherein R2 can include the groups:

and wherein Tr is a removable protecting group, for example a trityl,monomethoxytrityl, or dimethoxytrityl; n is an integer from about 1 toabout 50; and R8 is a nitrogen protecting group, for example aphthaloyl, trifluoroacetyl, FMOC, or monomethoxytrityl group.

In another embodiment, the invention features a compound having Formula61:

wherein X comprises a siNA molecule or portion thereof; each Windependently comprises a linker molecule or chemical linkage that canbe the same or different and can be present or absent, Y comprises alinker molecule that can be present or absent; each 5 independentlycomprises a signal protein or peptide, for example Human serum albuminprotein, Antennapedia peptide, Kaposi fibroblast growth factor peptide,Caiman crocodylus Ig(5) light chain peptide, HIV envelope glycoproteingp41 peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; each R1, R2, R3, and R4independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and n is aninteger from about 1 to about 10. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula62:

wherein X comprises a siNA molecule or portion thereof; each 5independently comprises a signal protein or peptide, for example Humanserum albumin protein, Antennapedia peptide, Kaposi fibroblast growthfactor peptide, Caiman crocodylus Ig(5) light chain peptide, HIVenvelope glycoprotein gp41 peptide, HIV-1 Tat peptide, Influenzahemagglutinin envelope glycoprotein peptide, or transportan A peptide; Wcomprises a linker molecule or chemical linkage that can be present orabsent; each R1, R2, and R3 independently comprises O, OH, H, alkyl,alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and each n is independently an integer from about 1 toabout 10. In another embodiment, W is selected from the group consistingof amide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

In another embodiment, the invention features a compound having Formula63:

wherein X comprises a siNA molecule or portion thereof; V comprises asignal protein or peptide, for example Human serum albumin protein,Antennapedia peptide, Kaposi fibroblast growth factor peptide, Caimancrocodylus Ig(5) light chain peptide, HIV envelope glycoprotein gp41peptide, HIV-1 Tat peptide, Influenza hemagglutinin envelopeglycoprotein peptide, or transportan A peptide; W comprises a linkermolecule or chemical linkage that can be present or absent; each R1, R2,R3 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, R4represents an ester, amide, or protecting group, and each n isindependently an integer from about 1 to about 10. In anotherembodiment, W is selected from the group consisting of amide, phosphate,phosphate ester, phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula64:

wherein X comprises a siNA molecule or portion thereof; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, A comprises a nitrogen containing group, and B comprisesa lipophilic group. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula65:

wherein X comprises a siNA molecule or portion thereof; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, RV comprises the lipid or phospholipid component of anyof Formulae 47-50, and R6 comprises a nitrogen containing group. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In another embodiment, the invention features a compound having Formula92:

wherein B comprises H, a nucleoside base, or a non-nucleosidic base withor without protecting groups; each R1 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, or aphosphorus containing group; X comprises H, a removable protectinggroup, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, enzymatic nucleic acid, amino acid, peptide, protein,lipid, phospholipid, biologically active molecule or label; W comprisesa linker molecule or chemical linkage that can be present or absent; R2comprises O, NH, S, CO, COO, ON═C, or alkyl; R3 comprises alkyl, akloxy,or an aminoacyl side chain; and SG comprises a sugar, for examplegalactose, galactosamine, N-acetyl-galactosamine, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers.

In another embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In another embodiment, the invention features a compound having Formula86:

wherein R1 comprises H, alkyl, alkylhalo, N, substituted N, or aphosphorus containing group; R2 comprises H, O, OH, alkyl, alkylhalo,halo, S, N, substituted N, or a phosphorus containing group; X comprisesH, a removable protecting group, a siNA molecule or a portion thereof; Wcomprises a linker molecule or chemical linkage that can be present orabsent; R3 comprises O, NH, S, CO, COO, ON═C, or alkyl; R4 comprisesalkyl, akloxy, or an aminoacyl side chain; and SG comprises a sugar, forexample galactose, galactosamine, N-acetyl-galactosamine, glucose,mannose, fructose, or fucose and the respective D or L, alpha or betaisomers, and each n is independently an integer from about 0 to about20. In another embodiment, W is selected from the group consisting ofamide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

In another embodiment, the invention features a compound having Formula87:

wherein X comprises a protein, peptide, antibody, lipid, phospholipid,oligosaccharide, label, biologically active molecule, for example avitamin such as folate, vitamin A, E, B6, B12, coenzyme, antibiotic,antiviral, nucleic acid, nucleotide, nucleoside, or oligonucleotide suchas an enzymatic nucleic acid, allozyme, antisense nucleic acid, siNA,2,5-A chimera, decoy, aptamer or triplex forming oligonucleotide, orpolymers such as polyethylene glycol; W comprises a linker molecule orchemical linkage that can be present or absent; and Y comprises siNA ora portion thereof; R1 comprises H, alkyl, or substituted alkyl. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In another embodiment, the invention features a compound having Formula88:

wherein X comprises a protein, peptide, antibody, lipid, phospholipid,oligosaccharide, label, biologically active molecule, for example avitamin such as folate, vitamin A, E, B6, B12, coenzyme, antibiotic,antiviral, nucleic acid, nucleotide, nucleoside, or oligonucleotide suchas an enzymatic nucleic acid, allozyme, antisense nucleic acid, siNA,2,5-A chimera, decoy, aptamer or triplex forming oligonucleotide, orpolymers such as polyethylene glycol; W comprises a linker molecule orchemical linkage that can be present or absent, and Y comprises a siNAor a portion thereof. In another embodiment, W is selected from thegroup consisting of amide, phosphate, phosphate ester, phosphoramidate,or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula99:

wherein X comprises a siNA molecule or portion thereof; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and SG comprises a sugar, for example galactose,galactosamine, N-acetyl-galactosamine or branched derivative thereof,glucose, mannose, fructose, or fucose and the respective D or L, alphaor beta isomers. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula100:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule that can be present or absent; each R1, R2,R3, and R4 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, and SGcomprises a sugar, for example galactose, galactosamine,N-acetyl-galactosamine or branched derivative thereof, glucose, mannose,fructose, or fucose and the respective D or L, alpha or beta isomers. Inanother embodiment, W is selected from the group consisting of amide,phosphate, phosphate ester, phosphoramidate, or thiophosphate esterlinkage.

In one embodiment, the SG component of any compound having Formulae 99or 100 comprises a compound having Formula 101:

wherein Y comprises a linker molecule or chemical linkage that can bepresent or absent and each R7 independently comprises an acyl group thatcan be present or absent, for example a acetyl group.

In one embodiment, the W-SG component of a compound having Formulae 99comprises a compound having Formula 102:

wherein R2 comprises O, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo,S, N, substituted N, a protecting group, or another compound havingFormula 102; R1 independently H, OH, alkyl, substituted alkyl, or haloand each R7 independently comprises an acyl group that can be present orabsent, for example a acetyl group, and R3 comprises O or R3 in Formula99, and n is an integer from about 1 to about 20.

In one embodiment, the W-SG component of a compound having Formulae 99comprises a compound having Formula 103:

wherein R1 comprises H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N,substituted N, a protecting group, or another compound having Formula103; each R7 independently comprises an acyl group that can be presentor absent, for example a acetyl group, and R3 comprises H or R3 inFormula 99, and each n is independently an integer from about 1 to about20.

In one embodiment, the invention features a compound having Formula 104:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently comprises an acyl group that can be present or absent, forexample a acetyl group, and each n is independently an integer fromabout 1 to about 20, and

wherein R1 can include the groups:

and wherein R2 can include the groups:

In one embodiment, the invention features a compound having Formula 105:

wherein X comprises a siNA molecule or a portion thereof, R2 comprisesO, OH, H, alkyl, alkylhalo, O-alkyl, O-alkylhalo, S, N, substituted N, aprotecting group, or a nucleotide, polynucleotide, or oligonucleotide ora portion thereof; R1 independently H, OH, alkyl, substituted alkyl, orhalo and each R7 independently comprises an acyl group that can bepresent or absent, for example a acetyl group, and n is an integer fromabout 1 to about 20.

In one embodiment, the invention features a compound having Formula 106:

wherein X comprises a siNA molecule or a portion thereof, R1 comprisesH, OH, amino, substituted amino, nucleotide, nucleoside, nucleic acid,oligonucleotide, amino acid, peptide, protein, lipid, phospholipid,label, or a portion thereof, or OR5 where R5 a removable protectinggroup, each R7 independently comprises an acyl group that can be presentor absent, for example a acetyl group, and each n is independently aninteger from about 1 to about 20

In another embodiment, the invention features a compound having Formula107:

wherein X comprises a siNA molecule or portion thereof; each Windependently comprises a linker molecule or chemical linkage that canbe present or absent, Y comprises a linker molecule that can be presentor absent; each R1, R2, R3, and R4 independently comprises O, OH, H,alkyl, alkylhalo, O-alkyl, O-alkylcyano, S, S-alkyl, S-alkylcyano, N orsubstituted N, and Cholesterol comprises cholesterol or an analog,derivative, or metabolite thereof. In another embodiment, W is selectedfrom the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In another embodiment, the invention features a compound having Formula108:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, Ycomprises a linker molecule that can be present or absent; each R1, R2,R3, and R4 independently comprises O, OH, H, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, andCholesterol comprises cholesterol or an analog, derivative, ormetabolite thereof. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In one embodiment, the W-Cholesterol component of a compound havingFormula 107 comprises a compound having Formula 109:

wherein R3 comprises R3 as described in Formula 107, and n isindependently an integer from about 1 to about 20.

In one embodiment, the invention features a compound having Formula 110:

wherein R4 comprises O, alkyl, alkylhalo, O-alkyl, O-alkylcyano, S,S-alkyl, S-alkylcyano, N or substituted N, each n is independently aninteger from about 1 to about 20, and

wherein R1 can include the groups:

and wherein R2 can include the groups:

In one embodiment, the invention features a compound having Formula 111:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, and nis an integer from about 1 to about 20. In another embodiment, W isselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 112:

wherein n is an integer from about 1 to about 20. In another embodiment,a compound having Formula 112 is used to generate a compound havingFormula 111 via NHS ester mediated coupling with a biologically activemolecule, such as a siNA molecule or a portion thereof. In anon-limiting example, the NHS ester coupling can be effectuated viaattachment to a free amine present in the siNA molecule, such as anamino linker molecule present on a nucleic acid sugar (e.g. 2′-aminolinker) or base (e.g., C5 alkyl amine linker) component of the siNAmolecule.

In one embodiment, the invention features a compound having Formula 113:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each n isindependently an integer from about 1 to about 20, and

wherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 113 is used to generatea compound having Formula 111 via phosphoramidite mediated coupling witha biologically active molecule, such as a siNA molecule or a portionthereof.

In one embodiment, the invention features a compound having Formula 114:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, and nis an integer from about 1 to about 20. In another embodiment, W isselected from the group consisting of amide, phosphate, phosphate ester,phosphoramidate, or thiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 115:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, R3comprises H, OH, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, label, or a portion thereof, or OR5 where R5 a removableprotecting group, and each n is independently an integer from about 1 toabout 20. In another embodiment, W is selected from the group consistingof amide, phosphate, phosphate ester, phosphoramidate, or thiophosphateester linkage.

In one embodiment, the invention features a compound having Formula 116:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each n isindependently an integer from about 1 to about 20, and

wherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 116 is used to generatea compound having Formula 114 or 115 via phosphoramidite mediatedcoupling with a biologically active molecule, such as a siNA molecule ora portion thereof.

In one embodiment, the invention features a compound having Formula 117:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently comprises an acyl group that can be present or absent, forexample a acetyl group, each n is independently an integer from about 1to about 20, and

wherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 117 is used to generatea compound having Formula 105 via phosphoramidite mediated coupling witha biologically active molecule, such as a siNA molecule or a portionthereof.

In one embodiment, the invention features a compound having Formula 118:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, R3comprises H, OH, amino, substituted amino, nucleotide, nucleoside,nucleic acid, oligonucleotide, amino acid, peptide, protein, lipid,phospholipid, label, or a portion thereof, or OR5 where R5 a removableprotecting group, each R7 independently comprises an acyl group that canbe present or absent, for example a acetyl group, and each n isindependently an integer from about 1 to about 20. In anotherembodiment, W is selected from the group consisting of amide, phosphate,phosphate ester, phosphoramidate, or thiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 119:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, eachR7 independently comprises an acyl group that can be present or absent,for example a acetyl group, and each n is independently an integer fromabout 1 to about 20. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 120:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently comprises an acyl group that can be present or absent, forexample a acetyl group, each n is independently an integer from about 1to about 20, and

wherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 120 is used to generatea compound having Formula 118 or 119 via phosphoramidite mediatedcoupling with a biologically active molecule, such as a siNA molecule ora portion thereof.

In one embodiment, the invention features a compound having Formula 121:

wherein X comprises a siNA molecule or portion thereof; W comprises alinker molecule or chemical linkage that can be present or absent, eachR7 independently comprises an acyl group that can be present or absent,for example a acetyl group, and each n is independently an integer fromabout 1 to about 20. In another embodiment, W is selected from the groupconsisting of amide, phosphate, phosphate ester, phosphoramidate, orthiophosphate ester linkage.

In one embodiment, the invention features a compound having Formula 122:

wherein R3 comprises H, OH, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, label, or a portion thereof, or OR5 where R5 aremovable protecting group, R4 comprises O, alkyl, alkylhalo, O-alkyl,O-alkylcyano, S, S-alkyl, S-alkylcyano, N or substituted N, each R7independently comprises an acyl group that can be present or absent, forexample a acetyl group, each n is independently an integer from about 1to about 20, and

wherein R1 can include the groups:

and wherein R2 can include the groups:

In another embodiment, a compound having Formula 122 is used to generatea compound having Formula 121 via phosphoramidite mediated coupling witha biologically active molecule, such as a siNA molecule or a portionthereof.

In one embodiment, the invention features a compound having Formula 94,X—Y—W—Y—Z   94

wherein X comprises a siNA molecule or a portion thereof; each Yindependently comprises a linker or chemical linkage that can be presentor absent, W comprises a biodegradable nucleic acid linker molecule, andZ comprises a biologically active molecule, for example an enzymaticnucleic acid, allozyme, antisense nucleic acid, siNA, 2,5-A chimera,decoy, aptamer or triplex forming oligonucleotide, peptide, protein, orantibody.

In another embodiment, W of a compound having Formula 94 of theinvention comprises 5′-cytidine-deoxythymidine-3′,5′-deoxythymidine-cytidine-3′, 5′-cytidine-deoxyuridine-3′,5′-deoxyuridine-cytidine-3′, 5′-uridine-deoxythymidine-3′, or5′-deoxythymidine-uridine-3′.

In yet another embodiment, W of a compound having Formula 94 of theinvention comprises 5′-adenosine-deoxythymidine-3′,5′-deoxythymidine-adenosine-3′, 5′-adenosine-deoxyuridine-3′, or5′-deoxyuridine-adenosine-3′.

In another embodiment, Y of a compound having Formula 94 of theinvention comprises a phosphorus containing linkage, phosphoramidatelinkage, phosphodiester linkage, phosphorothioate linkage, amidelinkage, ester linkage, carbamate linkage, disulfide linkage, oximelinkage, or morpholino linkage.

In another embodiment, compounds having Formula 89 and 91 of theinvention are synthesized by periodate oxidation of an N-terminal Serineor Threonine residue of a peptide or protein.

In one embodiment, X of compounds having Formulae 43, 44, 46-52, 58,61-65, 85-88, 92, 94, 95, 99, 100, 105-108, 111, 114, 115, 118, 119, or121 of the invention comprises a siNA molecule or a portion thereof. Inone embodiment, the siNA molecule can be conjugated at the 5′ end,3′-end, or both 5′ and 3′ ends of the sense strand or region of thesiNA. In one embodiment, the siNA molecule can be conjugated at the3′-end of the antisense strand or region of the siNA with a compound ofthe invention. In one embodiment, both the sense strand and antisensestrands or regions of the siNA molecule are conjugated with a compoundof the invention. In one embodiment, only the sense strand or region ofthe siNA is conjugated with a compound of the invention. In oneembodiment, only the antisense strand or region of the siNA isconjugated with a compound of the invention.

In one embodiment, W and/or Y of compounds having Formulae 43, 44,46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114,115, 118, 119, or 121 of the invention comprises a degradable orcleavable linker, for example a nucleic acid sequence comprisingribonucleotides and/or deoxynucleotides, such as a dimer, trimer, ortetramer. A non limiting example of a nucleic acid cleavable linker isan adenosine-deoxythymidine (A-dT) dimer or a cytidine-deoxythymidine(C-dT) dimer. In yet another embodiment, W and/or V of compounds havingFormulae 43, 44, 48-51, 58, 63-65, 96, 99, 100, 107, 108, 111, 114, 115,118, 119, or 121 of the invention comprises a N-hydroxy succinimide(NHS) ester linkage, oxime linkage, disulfide linkage, phosphoramidate,phosphorothioate, phosphorodithioate, phosphodiester linkage, or NHC(O),CH₃NC(O), CONH, C(O)NCH₃, S, SO, SO₂, O, NH, NCH₃ group. In anotherembodiment, the degradable linker, W and/or Y, of compounds havingFormulae Formulae 43, 44, 46-52, 58, 61-65, 85-88, 92, 94, 95, 99, 100,101, 107, 108, 111, 114, 115, 118, 119, or 121 of the inventioncomprises a linker that is susceptible to cleavage by carboxypeptidaseactivity.

In another embodiment, W and/or Y of Formulae 43, 44, 46-52, 58, 61-65,85-88, 92, 94, 95, 99, 100, 101, 107, 108, 111, 114, 115, 118, 119, or121 comprises a polyethylene glycol linker having Formula 45:

wherein Z comprises H, OH, O-alkyl, SH, S-alkyl, alkyl, substitutedalkyl, aryl, substituted aryl, amino, substituted amino, nucleotide,nucleoside, nucleic acid, oligonucleotide, amino acid, peptide, protein,lipid, phospholipid, or label; and n is an integer from about 1 to about100.

In one embodiment, the nucleic acid conjugates of the instant inventionare assembled by solid phase synthesis, for example on an automatedpeptide synthesizer, for example a Miligen 9050 synthesizer and/or anautomated oligonucleotide synthesizer such as an ABI 394, 390Z, orPharmacia OligoProcess, OligoPilot, OligoMax, or AKTA synthesizer. Inanother embodiment, the nucleic acid conjugates of the invention areassembled post synthetically, for example, following solid phaseoligonucleotide synthesis (see for example FIGS. 45, 50, 53, and 73).

In another embodiment, V of compounds having Formula 58-63 and 96comprise peptides having SEQ ID NOS: 507-516 (Table V).

In one embodiment, the nucleic acid conjugates of the instant inventionare assembled post synthetically, for example, following solid phaseoligonucleotide synthesis.

The present invention provides compositions and conjugates comprisingnucleosidic and non-nucleosidic derivatives. The present invention alsoprovides nucleic acid, polynucleotide and oligonucleotide derivativesincluding RNA, DNA, and PNA based conjugates. The attachment ofcompounds of the invention to nucleosides, nucleotides, non-nucleosides,and nucleic acid molecules is provided at any position within themolecule, for example, at internucleotide linkages, nucleosidic sugarhydroxyl groups such as 5′, 3′, and 2′-hydroxyls, and/or at nucleobasepositions such as amino and carbonyl groups.

The exemplary conjugates of the invention are described as compounds ofthe formulae herein, however, other peptide, protein, phospholipid, andpoly-alkyl glycol derivatives are provided by the invention, includingvarious analogs of the compounds of formulae 1-122, including but notlimited to different isomers of the compounds described herein.

The exemplary folate conjugates of the invention are described ascompounds shown by formulae herein, however, other folate and antifolatederivatives are provided by the invention, including various folateanalogs of the formulae of the invention, including dihydrofloates,tetrahydrofolates, tetrahydorpterins, folinic acid, pteropolyglutamicacid, 1-deaza, 3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10dideaza, 8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroicacids. As used herein, the term “folate” is meant to refer to folate andfolate derivatives, including pteroic acid derivatives and analogs.

The present invention features compositions and conjugates to facilitatedelivery of molecules into a biological system such as cells. Theconjugates provided by the instant invention can impart therapeuticactivity by transferring therapeutic compounds across cellularmembranes. The present invention encompasses the design and synthesis ofnovel agents for the delivery of molecules, including but not limited tosiNA molecules. In general, the transporters described are designed tobe used either individually or as part of a multi-component system. Thecompounds of the invention generally shown in Formulae herein areexpected to improve delivery of molecules into a number of cell typesoriginating from different tissues, in the presence or absence of serum.

In another embodiment, the compounds of the invention are provided as asurface component of a lipid aggregate, such as a liposome encapsulatedwith the predetermined molecule to be delivered. Liposomes, which can beunilamellar or multilamellar, can introduce encapsulated material into acell by different mechanisms. For example, the liposome can directlyintroduce its encapsulated material into the cell cytoplasm by fusingwith the cell membrane. Alternatively, the liposome can becompartmentalized into an acidic vacuole (i.e., an endosome) and itscontents released from the liposome and out of the acidic vacuole intothe cellular cytoplasm.

In one embodiment the invention features a lipid aggregate formulationof the compounds described herein, including phosphatidylcholine (ofvarying chain length; e.g., egg yolk phosphatidylcholine), cholesterol,a cationic lipid, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethyleneglycol-2000(DSPE-PEG2000). The cationic lipid component of this lipid aggregate canbe any cationic lipid known in the art such as dioleoyl1,2,-diacyl-3-trimethylammonium-propane (DOTAP). In another embodimentthis cationic lipid aggregate comprises a covalently bound compounddescribed in any of the Formulae herein.

In another embodiment, polyethylene glycol (PEG) is covalently attachedto the compounds of the present invention. The attached PEG can be anymolecular weight but is preferably between 2000-50,000 daltons.

The compounds and methods of the present invention are useful forintroducing nucleotides, nucleosides, nucleic acid molecules, lipids,peptides, proteins, and/or non-nucleosidic small molecules into a cell.For example, the invention can be used for nucleotide, nucleoside,nucleic acid, lipids, peptides, proteins, and/or non-nucleosidic smallmolecule delivery where the corresponding target site of action existsintracellularly.

In one embodiment, the compounds of the instant invention provideconjugates of molecules that can interact with cellular receptors, suchas high affinity folate receptors and ASGPr receptors, and provide anumber of features that allow the efficient delivery and subsequentrelease of conjugated compounds across biological membranes. Thecompounds utilize chemical linkages between the receptor ligand and thecompound to be delivered of length that can interact preferentially withcellular receptors. Furthermore, the chemical linkages between theligand and the compound to be delivered can be designed as degradablelinkages, for example by utilizing a phosphate linkage that is proximalto a nucleophile, such as a hydroxyl group. Deprotonation of thehydroxyl group or an equivalent group, as a result of pH or interactionwith a nuclease, can result in nucleophilic attack of the phosphateresulting in a cyclic phosphate intermediate that can be hydrolyzed.This cleavage mechanism is analogous RNA cleavage in the presence of abase or RNA nuclease. Alternately, other degradable linkages can beselected that respond to various factors such as UV irradiation,cellular nucleases, pH, temperature etc. The use of degradable linkagesallows the delivered compound to be released in a predetermined system,for example in the cytoplasm of a cell, or in a particular cellularorganelle.

The present invention also provides ligand derived phosphoramidites thatare readily conjugated to compounds and molecules of interest.Phosphoramidite compounds of the invention permit the direct attachmentof conjugates to molecules of interest without the need for usingnucleic acid phosphoramidite species as scaffolds. As such, the used ofphosphoramidite chemistry can be used directly in coupling the compoundsof the invention to a compound of interest, without the need for othercondensation reactions, such as condensation of the ligand to an aminogroup on the nucleic acid, for example at the N6 position of adenosineor a 2′-deoxy-2′-amino function. Additionally, compounds of theinvention can be used to introduce non-nucleic acid based conjugatedlinkages into oligonucleotides that can provide more efficient couplingduring oligonucleotide synthesis than the use of nucleic acid-basedphosphoramidites. This improved coupling can take into account improvedsteric considerations of abasic or non-nucleosidic scaffolds bearingpendant alkyl linkages.

Compounds of the invention utilizing triphosphate groups can be utilizedin the enzymatic incorporation of conjugate molecules intooligonucleotides. Such enzymatic incorporation is useful when conjugatesare used in post-synthetic enzymatic conjugation or selection reactions,(see for example Matulic-Adamic et al., 2000, Bioorg. Med. Chem. Lett.,10, 1299-1302; Lee et al., 2001, NAR., 29, 1565-1573; Joyce, 1989, Gene,82, 83-87; Beaudry et al., 1992, Science 257, 635-641; Joyce, 1992,Scientific American 267, 90-97; Breaker et al., 1994, TIBTECH 12, 268;Bartel et al., 1993, Science 261:1411-1418; Szostak, 1993, TIBS 17,89-93; Kumar et al., 1995, FASEB J., 9, 1183; Breaker, 1996, Curr. Op.Biotech., 7, 442; Santoro et al., 1997, Proc. Natl. Acad. Sci., 94,4262; Tang et al., 1997, RNA 3, 914; Nakamaye & Eckstein, 1994, supra;Long & Uhlenbeck, 1994, supra; Ishizaka et al., 1995, supra; Vaish etal., 1997, Biochemistry 36, 6495; Kuwabara et al., 2000, Curr. Opin.Chem. Biol., 4, 669).

The term “biodegradable linker” as used herein, refers to a nucleic acidor non-nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siNA molecule of the invention or thesense and antisense strands of a siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein, refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

The term “alkyl” as used herein refers to a saturated aliphatichydrocarbon, including straight-chain, branched-chain “isoalkyl”, andcyclic alkyl groups. The term “alkyl” also comprises alkoxy, alkyl-thio,alkyl-thio-alkyl, alkoxyalkyl, alkylamino, alkenyl, alkynyl, alkoxy,cycloalkenyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl,C1-C6 hydrocarbyl, aryl or substituted aryl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably it is a lower alkyl offrom about 1 to about 7 carbons, more preferably about 1 to about 4carbons. The alkyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. The term “alkyl” alsoincludes alkenyl groups containing at least one carbon-carbon doublebond, including straight-chain, branched-chain, and cyclic groups.Preferably, the alkenyl group has about 2 to about 12 carbons. Morepreferably it is a lower alkenyl of from about 2 to about 7 carbons,more preferably about 2 to about 4 carbons. The alkenyl group can besubstituted or unsubstituted. When substituted the substituted group(s)preferably comprise hydroxy, oxy, thio, amino, nitro, cyano, alkoxy,alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, silyl, alkenyl,alkynyl, alkoxy, cycloalkenyl, cycloalkyl, cycloalkylalkyl,heterocycloalkyl, heteroaryl, C1-C6 hydrocarbyl, aryl or substitutedaryl groups. The term “alkyl” also includes alkynyl groups containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group hasabout 2 to about 12 carbons. More preferably it is a lower alkynyl offrom about 2 to about 7 carbons, more preferably about 2 to about 4carbons. The alkynyl group can be substituted or unsubstituted. Whensubstituted the substituted group(s) preferably comprise hydroxy, oxy,thio, amino, nitro, cyano, alkoxy, alkyl-thio, alkyl-thio-alkyl,alkoxyalkyl, alkylamino, silyl, alkenyl, alkynyl, alkoxy, cycloalkenyl,cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, C1-C6hydrocarbyl, aryl or substituted aryl groups. Alkyl groups or moietiesof the invention can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. The preferred substituent(s)of aryl groups are halogen, trihalomethyl, hydroxyl, SH, OH, cyano,alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An “alkylaryl” grouprefers to an alkyl group (as described above) covalently joined to anaryl group (as described above). Carbocyclic aryl groups are groupswherein the ring atoms on the aromatic ring are all carbon atoms. Thecarbon atoms are optionally substituted. Heterocyclic aryl groups aregroups having from about 1 to about 3 heteroatoms as ring atoms in thearomatic ring and the remainder of the ring atoms are carbon atoms.

Suitable heteroatoms include oxygen, sulfur, and nitrogen, and includefuranyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo, pyrimidyl,pyrazinyl, imidazolyl and the like, all optionally substituted. An“amide” refers to an —C(O)—NH—R, where R is either alkyl, aryl,alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′, where R iseither alkyl, aryl, alkylaryl or hydrogen.

The term “alkoxyalkyl” as used herein refers to an alkyl-O-alkyl ether,for example, methoxyethyl or ethoxymethyl.

The term “alkyl-thio-alkyl” as used herein refers to an alkyl-S-alkylthioether, for example, methylthiomethyl or methylthioethyl.

The term “amino” as used herein refers to a nitrogen containing group asis known in the art derived from ammonia by the replacement of one ormore hydrogen radicals by organic radicals. For example, the terms“aminoacyl” and “aminoalkyl” refer to specific N-substituted organicradicals with acyl and alkyl substituent groups respectively.

The term “amination” as used herein refers to a process in which anamino group or substituted amine is introduced into an organic molecule.

The term “exocyclic amine protecting moiety” as used herein refers to anucleobase amino protecting group compatible with oligonucleotidesynthesis, for example, an acyl or amide group.

The term “alkenyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon double bond. Examples of “alkenyl” include vinyl, allyl,and 2-methyl-3-heptene.

The term “alkoxy” as used herein refers to an alkyl group of indicatednumber of carbon atoms attached to the parent molecular moiety throughan oxygen bridge. Examples of alkoxy groups include, for example,methoxy, ethoxy, propoxy and isopropoxy.

The term “alkynyl” as used herein refers to a straight or branchedhydrocarbon of a designed number of carbon atoms containing at least onecarbon-carbon triple bond. Examples of “alkynyl” include propargyl,propyne, and 3-hexyne.

The term “aryl” as used herein refers to an aromatic hydrocarbon ringsystem containing at least one aromatic ring. The aromatic ring canoptionally be fused or otherwise attached to other aromatic hydrocarbonrings or non-aromatic hydrocarbon rings. Examples of aryl groupsinclude, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthaleneand biphenyl. Preferred examples of aryl groups include phenyl andnaphthyl.

The term “cycloalkenyl” as used herein refers to a C3-C8 cyclichydrocarbon containing at least one carbon-carbon double bond. Examplesof cycloalkenyl include cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadiene, cyclohexenyl, 1,3-cyclohexadiene, cycloheptenyl,cycloheptatrienyl, and cyclooctenyl.

The term “cycloalkyl” as used herein refers to a C3-C8 cyclichydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

The term “cycloalkylalkyl,” as used herein, refers to a C3-C7 cycloalkylgroup attached to the parent molecular moiety through an alkyl group, asdefined above. Examples of cycloalkylalkyl groups includecyclopropylmethyl and cyclopentylethyl.

The terms “halogen” or “halo” as used herein refers to indicatefluorine, chlorine, bromine, and iodine.

The term “heterocycloalkyl,” as used herein refers to a non-aromaticring system containing at least one heteroatom selected from nitrogen,oxygen, and sulfur. The heterocycloalkyl ring can be optionally fused toor otherwise attached to other heterocycloalkyl rings and/ornon-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups havefrom 3 to 7 members. Examples of heterocycloalkyl groups include, forexample, piperazine, morpholine, piperidine, tetrahydrofuran,pyrrolidine, and pyrazole. Preferred heterocycloalkyl groups includepiperidinyl, piperazinyl, morpholinyl, and pyrolidinyl.

The term “heteroaryl” as used herein refers to an aromatic ring systemcontaining at least one heteroatom selected from nitrogen, oxygen, andsulfur. The heteroaryl ring can be fused or otherwise attached to one ormore heteroaryl rings, aromatic or non-aromatic hydrocarbon rings orheterocycloalkyl rings. Examples of heteroaryl groups include, forexample, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline andpyrimidine. Preferred examples of heteroaryl groups include thienyl,benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl,benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl,isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl,tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.

The term “C1-C6 hydrocarbyl” as used herein refers to straight,branched, or cyclic alkyl groups having 1-6 carbon atoms, optionallycontaining one or more carbon-carbon double or triple bonds. Examples ofhydrocarbyl groups include, for example, methyl, ethyl, propyl,isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, isopentyl,neopentyl, hexyl, 2-hexyl, 3-hexyl, 3-methylpentyl, vinyl, 2-pentene,cyclopropylmethyl, cyclopropyl, cyclohexylmethyl, cyclohexyl andpropargyl. When reference is made herein to C1-C6 hydrocarbyl containingone or two double or triple bonds it is understood that at least twocarbons are present in the alkyl for one double or triple bond, and atleast four carbons for two double or triple bonds.

The term “protecting group” as used herein, refers to groups known inthe art that are readily introduced and removed from an atom, forexample O, N, P, or S. Protecting groups are used to prevent undesirablereactions from taking place that can compete with the formation of aspecific compound or intermediate of interest. See also “ProtectiveGroups in Organic Synthesis”, 3rd Ed., 1999, Greene, T. W. and relatedpublications.

The term “nitrogen protecting group,” as used herein, refers to groupsknown in the art that are readily introduced on to and removed from anitrogen. Examples of nitrogen protecting groups include Boc, Cbz,benzoyl, and benzyl. See also “Protective Groups in Organic Synthesis”,3rd Ed., 1999, Greene, T. W. and related publications.

The term “hydroxy protecting group,” or “hydroxy protection” as usedherein, refers to groups known in the art that are readily introduced onto and removed from an oxygen, specifically an —OH group. Examples ofhyroxy protecting groups include trityl or substituted trityl groups,such as monomethoxytrityl and dimethoxytrityl, or substituted silylgroups, such as tert-butyldimethyl, trimethylsilyl, ortert-butyldiphenyl silyl groups. See also “Protective Groups in OrganicSynthesis”, 3rd Ed., 1999, Greene, T. W. and related publications.

The term “acyl” as used herein refers to —C(O)R groups, wherein R is analkyl or aryl.

The term “phosphorus containing group” as used herein, refers to achemical group containing a phosphorus atom. The phosphorus atom can betrivalent or pentavalent, and can be substituted with O, H, N, S, C orhalogen atoms. Examples of phosphorus containing groups of the instantinvention include but are not limited to phosphorus atoms substitutedwith O, H, N, S, C or halogen atoms, comprising phosphonate,alkylphosphonate, phosphate, diphosphate, triphosphate, pyrophosphate,phosphorothioate, phosphorodithioate, phosphoramidate, phosphoramiditegroups, nucleotides and nucleic acid molecules.

The term “phosphine” or “phosphite” as used herein refers to a trivalentphosphorus species, for example compounds having Formula 97:

-   -   wherein R can include the groups:

-   -   and wherein S and T independently include the groups:

The term “phosphate” as used herein refers to a pentavalent phosphorusspecies, for example a compound having Formula 98:

-   -   wherein R includes the groups:

and wherein S and T each independently can be a sulfur or oxygen atom ora group which can include:

and wherein M comprises a sulfur or oxygen atom. The phosphate of theinvention can comprise a nucleotide phosphate, wherein any R, S, or T inFormula 98 comprises a linkage to a nucleic acid or nucleoside.

The term “cationic salt” as used herein refers to any organic orinorganic salt having a net positive charge, for example atriethylammonium (TEA) salt.

The term “degradable linker” as used herein, refers to linker moietiesthat are capable of cleavage under various conditions. Conditionssuitable for cleavage can include but are not limited to pH, UVirradiation, enzymatic activity, temperature, hydrolysis, elimination,and substitution reactions, and thermodynamic properties of the linkage.

The term “photolabile linker” as used herein, refers to linker moietiesas are known in the art, that are selectively cleaved under particularUV wavelengths. Compounds of the invention containing photolabilelinkers can be used to deliver compounds to a target cell or tissue ofinterest, and can be subsequently released in the presence of a UVsource.

The term “nucleic acid conjugates” as used herein, refers to nucleoside,nucleotide and oligonucleotide conjugates.

The term “lipid” as used herein, refers to any lipophilic compound.Non-limiting examples of lipid compounds include fatty acids and theirderivatives, including straight chain, branched chain, saturated andunsaturated fatty acids, carotenoids, terpenes, bile acids, andsteroids, including cholesterol and derivatives or analogs thereof.

The term “folate” as used herein, refers to analogs and derivatives offolic acid, for example antifolates, dihydrofloates, tetrahydrofolates,tetrahydorpterins, folinic acid, pteropolyglutamic acid, 1-deaza,3-deaza, 5-deaza, 8-deaza, 10-deaza, 1,5-deaza, 5,10 dideaza,8,10-dideaza, and 5,8-dideaza folates, antifolates, and pteroic acidderivatives.

The term “compounds with neutral charge” as used herein, refers tocompositions which are neutral or uncharged at neutral or physiologicalpH. Examples of such compounds are cholesterol and other steroids,cholesteryl hemisuccinate (CHEMS), dioleoyl phosphatidyl choline,distearoylphosphotidyl choline (DSPC), fatty acids such as oleic acid,phosphatidic acid and its derivatives, phosphatidyl serine, polyethyleneglycol-conjugated phosphatidylamine, phosphatidylcholine,phosphatidylethanolamine and related variants, prenylated compoundsincluding farnesol, polyprenols, tocopherol, and their modified forms,diacylsuccinyl glycerols, fusogenic or pore forming peptides,dioleoylphosphotidylethanolamine (DOPE), ceramide and the like.

The term “lipid aggregate” as used herein refers to a lipid-containingcomposition wherein the lipid is in the form of a liposome, micelle(non-lamellar phase) or other aggregates with one or more lipids.

The term “nitrogen containing group” as used herein refers to anychemical group or moiety comprising a nitrogen or substituted nitrogen.Non-limiting examples of nitrogen containing groups include amines,substituted amines, amides, alkylamines, amino acids such as arginine orlysine, polyamines such as spermine or spermidine, cyclic amines such aspyridines, pyrimidines including uracil, thymine, and cytosine,morpholines, phthalimides, and heterocyclic amines such as purines,including guanine and adenine.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes; nucleic acid molecules coupled with known smallmolecule modulators; or intermittent treatment with combinations ofmolecules, including different motifs and/or other chemical orbiological molecules). The treatment of subjects with siNA molecules canalso include combinations of different types of nucleic acid molecules,such as enzymatic nucleic acid molecules (ribozymes), allozymes,antisense, 2,5-A oligoadenylate, decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more5′ and/or a 3′-cap structure, for example on only the sense siNA strand,the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and can help in delivery and/orlocalization within a cell. The cap can be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or can be present on bothtermini. Non-limiting examples of the 5′-cap include, but are notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′, 5′-methylenenucleotide; 1-(beta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphonoacetate, and/or thiophosphonoacetate,phosphorodithioate, methylphosphonate, phosphotriester, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl,substitutions. For a review of oligonucleotide backbone modifications,see Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis andProperties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker etal., 1994, Novel Backbone Replacements for Oligonucleotides, inCarbohydrate Modifications in Antisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, see for exampleAdamic et al., U.S. Pat. No. 5,998,203.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon of(3-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae I-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O— NH₂, which can bemodified or unmodified. Such modified groups are described, for example,in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al.,U.S. Pat. No. 6,248,878, which are both incorporated by reference intheir entireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to treat anydisease, infection or condition associated with gene expression, andother indications that can respond to the level of gene product in acell or tissue, alone or in combination with other therapies. Forexample, a siNA molecule can comprise a delivery vehicle, includingliposomes, for administration to a subject, carriers and diluents andtheir salts, and/or can be present in pharmaceutically acceptableformulations. Methods for the delivery of nucleic acid molecules aredescribed in Akhtar et al., 1992, Trends Cell Bio., 2, 139; DeliveryStrategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 1995,Maurer et al., 1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang,1999, Handb. Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACSSymp. Ser., 752, 184-192, all of which are incorporated herein byreference. Beigelman et al., U.S. Pat. No. 6,395,713 and Sullivan etal., PCT WO 94/02595 further describe the general methods for deliveryof nucleic acid molecules. These protocols can be utilized for thedelivery of virtually any nucleic acid molecule. Nucleic acid moleculescan be administered to cells by a variety of methods known to those ofskill in the art, including, but not restricted to, encapsulation inliposomes, by iontophoresis, or by incorporation into other vehicles,such as biodegradable polymers, hydrogels, cyclodextrins (see forexample Gonzalez et al., 1999, Bioconjugate Chem., 10, 1068-1074; Wanget al., International PCT publication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In one embodiment,nucleic acid molecules or the invention are administered viabiodegradable implant materials, such as elastic shape memory polymers(see for example Lendelein and Langer, 2002, Science, 296, 1673).Alternatively, the nucleic acid/vehicle combination is locally deliveredby direct injection or by use of an infusion pump. Direct injection ofthe nucleic acid molecules of the invention, whether subcutaneous,intramuscular, or intradermal, can take place using standard needle andsyringe methodologies, or by needle-free technologies such as thosedescribed in Conry et al., 1999, Clin. Cancer Res., 5, 2330-2337 andBarry et al., International PCT Publication No. WO 99/31262. Manyexamples in the art describe CNS delivery methods of oligonucleotides byosmotic pump, (see Chun et al., 1998, Neuroscience Letters, 257,135-138, D'Aldin et al., 1998, Mol. Brain Research, 55, 151-164, Drydenet al., 1998, J. Endocrinol., 157, 169-175, Ghirnikar et al., 1998,Neuroscience Letters, 247, 21-24) or direct infusion (Broaddus et al.,1997, Neurosurg. Focus, 3, article 4). Other routes of delivery include,but are not limited to oral (tablet or pill form) and/or intrathecaldelivery (Gold, 1997, Neuroscience, 76, 1153-1158). More detaileddescriptions of nucleic acid delivery and administration are provided inSullivan et al., supra, Draper et al., PCT WO93/23569, Beigelman et al.,PCT WO99/05094, and Klimuk et al., PCT WO99/04819 all of which have beenincorporated by reference herein. The molecules of the instant inventioncan be used as pharmaceutical agents. Pharmaceutical agents prevent,modulate the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state in a subject.

In addition, the invention features the use of methods to deliver thenucleic acid molecules of the instant invention to hematopoietic cells,including monocytes and lymphocytes. These methods are described indetail by Hartmann et al., 1998, J. Phamacol. Exp. Ther., 285(2),920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion andPhillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei,1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, NucleicAcids Research, 22(22), 4681-8. Such methods, as described above,include the use of free oligonucleotide, cationic lipid formulations,liposome formulations including pH sensitive liposomes andimmunoliposomes, and bioconjugates including oligonucleotides conjugatedto fusogenic peptides, for the transfection of hematopoietic cells witholigonucleotides.

In one embodiment, a compound, molecule, or composition for thetreatment of ocular conditions (e.g., macular degeneration, diabeticretinopathy etc.) is administered to a subject intraocularly or byintraocular means. In another embodiment, a compound, molecule, orcomposition for the treatment of ocular conditions (e.g., maculardegeneration, diabetic retinopathy etc.) is administered to a subjectperiocularly or by periocular means (see for example Ahlheim et al.,International PCT publication No. WO 03/24420). In one embodiment, asiNA molecule and/or formulation or composition thereof is administeredto a subject intraocularly or by intraocular means. In anotherembodiment, a siNA molecule and/or formulation or composition thereof isadministered to a subject periocularly or by periocular means.Periocular administration generally provides a less invasive approach toadministering siNA molecules and formulation or composition thereof to asubject (see for example Ahlheim et al., International PCT publicationNo. WO 03/24420). The use of periocular administraction also minimizesthe risk of retinal detachment, allows for more frequent dosing oradministraction, provides a clinically relevant route of administractionfor macular degeneration and other optic conditions, and also providesthe possibility of using reservoirs (e.g., implants, pumps or otherdevices) for drug delivery.

In one embodiment, a siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PhramaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of Gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. Nos. 6,528,631; 6,335,434; 6,235,886; 6,153,737; 5,214,136;5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedinto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as tablets, capsules orelixirs for oral administration, suppositories for rectaladministration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemicadministration, into a cell or subject, including for example a human.Suitable forms, in part, depend upon the use or the route of entry, forexample oral, transdermal, or by injection. Such forms should notprevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

By “systemic administration” is meant in vivo systemic absorption oraccumulation of drugs in the blood stream followed by distributionthroughout the entire body. Administration routes that lead to systemicabsorption include, without limitation: intravenous, subcutaneous,intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.Each of these administration routes exposes the siNA molecules of theinvention to an accessible diseased tissue. The rate of entry of a druginto the circulation has been shown to be a function of molecular weightor size. The use of a liposome or other drug carrier comprising thecompounds of the instant invention can potentially localize the drug,for example, in certain tissue types, such as the tissues of thereticular endothelial system (RES). A liposome formulation that canfacilitate the association of drug with the surface of cells, such as,lymphocytes and macrophages is also useful. This approach can provideenhanced delivery of the drug to target cells by taking advantage of thespecificity of macrophage and lymphocyte immune recognition of abnormalcells, such as cancer cells.

By “pharmaceutically acceptable formulation” is meant a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity. Non-limiting examples of agentssuitable for formulation with the nucleic acid molecules of the instantinvention include: P-glycoprotein inhibitors (such as Pluronic P85),which can enhance entry of drugs into the CNS (Jolliet-Riant andTillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery after intracerebral implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc. Cambridge,Mass.); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate, which can deliver drugs across the blood brainbarrier and can alter neuronal uptake mechanisms (ProgNeuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999). Othernon-limiting examples of delivery strategies for the nucleic acidmolecules of the instant invention include material described in Boadoet al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et al., 1999, FEBSLett., 421, 280-284; Pardridge et al., 1995, PNAS USA., 92, 5592-5596;Boado, 1995, Adv. Drug Delivery Rev., 15, 73-107; Aldrian-Herrada etal., 1998, Nucleic Acids Res., 26, 4910-4916; and Tyler et al., 1999,PNAS USA., 96, 7053-7058.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995,95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).Such liposomes have been shown to accumulate selectively in tumors,presumably by extravasation and capture in the neovascularized targettissues (Lasic et al., Science 1995, 267, 1275-1276; Oku et al., 1995,Biochim. Biophys. Acta, 1238, 86-90). The long-circulating liposomesenhance the pharmacokinetics and pharmacodynamics of DNA and RNA,particularly compared to conventional cationic liposomes which are knownto accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995,42, 24864-24870; Choi et al., International PCT Publication No. WO96/10391; Ansell et al., International PCT Publication No. WO 96/10390;Holland et al., International PCT Publication No. WO 96/10392).Long-circulating liposomes are also likely to protect drugs fromnuclease degradation to a greater extent compared to cationic liposomes,based on their ability to avoid accumulation in metabolically aggressiveMPS tissues such as the liver and spleen.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in-water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J. Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatenarry or monoatennary chains (Baenziger and Fiete, 1980, Cell, 22,611-620; Connolly et al., 1982, J. Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J. Med. Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavialability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 10/151,116, filed May 17, 2002. In one embodiment, nucleic acidmolecules of the invention are complexed with or covalently attached tonanoparticles, such as Hepatitis B virus S, M, or L evelope proteins(see for example Yamado et al., 2003, Nature Biotechnology, 21, 885). Inone embodiment, nucleic acid molecules of the invention are deliveredwith specificity for human tumor cells, specifically non-apoptotic humantumor cells including for example T-cells, hepatocytes, breast carcinomacells, ovarian carcinoma cells, melanoma cells, intestinal epithelialcells, prostate cells, testicular cells, non-small cell lung cancers,small cell lung cancers, etc.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1: Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example using a Waters C18 SepPak Ig cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H₂O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H₂O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H₂O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H₂O followed by 1 CV 1M NaCl and additional H₂O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2: Serum Stability of Chemically Modified siNA Constructs

Chemical modifications were introduced into siNA constructs to determinethe stability of these constructs compared to native siNAoligonucleotides (containing two thymidine nucleotide overhangs) inhuman serum. An investigation of the serum stability of RNA duplexesrevealed that siNA constructs consisting of all RNA nucleotidescontaining two thymidine nucleotide overhangs have a half-life in serumof 15 seconds, whereas chemically modified siNA constructs remainedstable in serum for 1 to 3 days depending on the extent of modification(see FIG. 3). RNAi stability tests were performed by internally labelingone strand (strand 1) of siNA and duplexing with 1.5× the concentrationof the complementary siNA strand (strand 2) (to insure all labeledmaterial was in duplex form). Duplexed siNA constructs were then testedfor stability by incubating at a final concentration of 2 M siNA (strand2 concentration) in 90% mouse or human serum for time-points of 30 sec,1 min, 5 min, 30 min, 90 min, 4 hrs 10 min, 16 hrs 24 min, and 49 hrs.Time points were run on a 15% denaturing polyacrylamide gels andanalyzed on a phosphoimager.

Internal labeling was performed via kinase reactions with polynucleotidekinase (PNK) and ³²P-γ-ATP, with addition of radiolabeled phosphate atnucleotide 13 of strand 2, counting in from the 3′ side. Ligation of theremaining 8-mer fragments with T4 RNA ligase resulted in the fulllength, 21-mer, strand 2. Duplexing of RNAi was done by addingappropriate concentrations of the siNA oligonucleotides and heating to95° C. for 5 minutes followed by slow cooling to room temperature.Reactions were performed by adding 100% serum to the siNA duplexes andincubating at 37° C., then removing aliquots at desired time-points.Results of this study are summarized in FIG. 3. As shown in the FIG. 3,chemically modified siNA molecules (e.g., SEQ ID NOs: 412/413, 412/414,412/415, 412/416, and 412/418) have significantly increased serumstability compared to an siNA construct having all ribonucleotidesexcept a 3′-terminal dithymidine (TT) modification (e.g., SEQ ID NOs:419/420).

Example 3: Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a viral or human mRNAtranscript, is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays or combinatorial/siNA library screeningassays to determine efficient reduction in target gene expression.

Example 4: Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

The target sequence is parsed in silico into a list of all fragments orsubsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

In some instances the siNAs correspond to more than one target sequence;such would be the case for example in targeting different transcripts ofthe same gene, targeting different transcripts of more than one gene, orfor targeting both the human gene and an animal homolog. In this case, asubsequence list of a particular length is generated for each of thetargets, and then the lists are compared to find matching sequences ineach list. The subsequences are then ranked according to the number oftarget sequences that contain the given subsequence; the goal is to findsubsequences that are present in most or all of the target sequences.Alternately, the ranking can identify subsequences that are unique to atarget sequence, such as a mutant target sequence. Such an approachwould enable the use of siNA to target specifically the mutant sequenceand not effect the expression of the normal sequence.

In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in case 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided other appropriately suitable sequences are available. CCC issearched in the target strand because that will place GGG in theantisense strand.

The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Tables I). If terminalTT residues are desired for the sequence (as described in paragraph 7),then the two 3′ terminal nucleotides of both the sense and antisensestrands are replaced by TT prior to synthesizing the oligos.

The siNA molecules are screened in an in vitro, cell culture or animalmodel system to identify the most active siNA molecule or the mostpreferred target site within the target RNA sequence.

In an alternate approach, a pool of siNA constructs specific to a targetsequence is used to screen for target sites in cells expressing targetRNA, such as human HeLa cells. The general strategy used in thisapproach is shown in FIGS. 21A-21D. A non-limiting example of such apool is a pool comprising sequences having antisense sequencescomplementary to the target RNA sequence and sense sequencescomplementary to the antisense sequences. Cells (e.g., HeLa cells)expressing the target gene are transfected with the pool of siNAconstructs and cells that demonstrate a phenotype associated with genesilencing are sorted. The pool of siNA constructs can be chemicallymodified as described herein and synthesized, for example, in a highthroughput manner. The siNA from cells demonstrating a positivephenotypic change (e.g., decreased target mRNA levels or target proteinexpression), are identified, for example by positional analysis withinthe assay, and are used to determine the most suitable target site(s)within the target RNA sequence based upon the complementary sequence tothe corresponding siNA antisense strand identified in the assay.

Example 5: RNAi Activity of Chemically Modified siNA Constructs

Short interfering nucleic acid (siNA) is emerging as a powerful tool forgene regulation. All-ribose siNA duplexes activate the RNAi pathway buthave limited utility as therapeutic compounds due to their nucleasesensitivity and short half-life in serum, as shown in Example 2 above.To develop nuclease-resistant siNA constructs for in vivo applications,siNAs that target luciferase mRNA and contain stabilizing chemicalmodifications were tested for activity in HeLa cells. The sequences forthe siNA oligonucleotide sequences used in this study are shown in TableI. Modifications included phosphorothioate linkages (P═S), 2′-O-methylnucleotides, or 2′-fluoro (F) nucleotides in one or both siNA strandsand various 3′-end stabilization chemistries, including 3′-glyceryl,3′-inverted abasic, 3′-inverted Thymidine, and/or Thymidine. The RNAiactivity of chemically stabilized siNA constructs was compared with theRNAi activity of control siNA constructs consisting of allribonucleotides at every position except the 3′-terminus which comprisedtwo thymidine nucleotide overhangs. Active siNA molecules containingstabilizing modifications such as described herein should prove usefulfor in vivo applications, given their enhanced nuclease-resistance.

A luciferase reporter system was utilized to test RNAi activity ofchemically modified siNA constructs compared to siNA constructsconsisting of all RNA nucleotides containing two thymidine nucleotideoverhangs. Sense and antisense siNA strands (20 uM each) were annealedby incubation in buffer (100 mM potassium acetate, 30 mM HEPES-KOH, pH7.4, 2 mM magnesium acetate) for 1 min. at 90° C. followed by 1 hour at37° C. Plasmids encoding firefly luciferase (pGL2) and renillaluciferase (pRLSV40) were purchased from Promega Biotech.

HeLa S3 cells were grown at 37° C. in DMEM with 5% FBS and seeded at15,300 cells in 100 ul media per well of a 96-well plate 24 hours priorto transfection. For transfection, 4 ul Lipofectamine 2000 (LifeTechnologies) was added to 96 ul OPTI-MEM, vortexed and incubated atroom temperature for 5 minutes. The 100 ul diluted lipid was then addedto a microtiter tube containing 5 ul pGL2 (200 ng/ul), 5 ul pRLSV40 (8ng/ul) 6 ul siNA (25 nM or 10 nM final), and 84 ul OPTI-MEM, vortexedbriefly and incubated at room temperature for 20 minutes. Thetransfection mix was then mixed briefly and 50 ul was added to each ofthree wells that contained HeLa S3 cells in 100 ul media. Cells wereincubated for 20 hours after transfection and analyzed for luciferaseexpression using the Dual luciferase assay according to themanufacturer's instructions (Promega Biotech). The results of this studyare summarized in FIGS. 4-16. The sequences of the siNA strands used inthis study are shown in Table I and are referred to by Sirna/RPI #in thefigures. Normalized luciferase activity is reported as the ratio offirefly luciferase activity to renilla luciferase activity in the samesample. Error bars represent standard deviation of triplicatetransfections. As shown in FIGS. 4-16, the RNAi activity of chemicallymodified constructs is often comparable to that of unmodified controlsiNA constructs, which consist of all ribonucleotides at every positionexcept the 3′-terminus which comprises two thymidine nucleotideoverhangs. In some instances, the RNAi activity of the chemicallymodified constructs is greater than the unmodified control siNAconstruct consisting of all ribonucleotides.

For example, FIG. 4 shows results obtained from a screen usingphosphorothioate modified siNA constructs. The Sirna/RPI 27654/27659construct contains phosphorothioate substitutions for every pyrimidinenucleotide in both sequences, the Sirna/RPI 27657/27662 constructcontains 5 terminal 3′-phosphorothioate substitutions in each strand,the Sirna/RPI 27649/27658 construct contains all phosphorothioatesubstitutions only in the antisense strand, whereas the Sirna/RPI27649/27660 and Sirna/RPI 27649/27661 constructs have unmodified sensestrands and varying degrees of phosphorothioate substitutions in theantisense strand. All of these constructs show significant RNAi activitywhen compared to a scrambled siNA control construct (27651/27652).

FIG. 5 shows results obtained from a screen using phosphorothioate(Sirna/RPI 28253/28255 and Sirna/RPI 28254/28256) and universal basesubstitutions (Sirna/RPI 28257/28259 and Sirna/RPI 28258/28260) comparedto the same controls described above, these modifications showequivalent or better RNAi activity when compared to the unmodifiedcontrol siNA construct.

FIG. 6 shows results obtained from a screen using 2′-O-methyl modifiedsiNA constructs in which the sense strand contains either 10 (Sirna/RPI28244/27650) or 5 (Sirna/RPI 28245/27650) 2′-O-methyl substitutions,both with comparable activity to the unmodified control siNA construct.

FIG. 7 shows results obtained from a screen using 2′-O-methyl or2′-deoxy-2′-fluoro modified siNA constructs compared to a controlconstruct consisting of all ribonucleotides at every position except the3′-terminus which comprises two thymidine nucleotide overhangs.

FIG. 8 compares a siNA construct containing six phosphorothioatesubstitutions in each strand (Sirna/RPI 28460/28461), where 5phosphorothioates are present at the 3′ end and a singlephosphorothioate is present at the 5′ end of each strand. This motifshows very similar activity to the control siNA construct consisting ofall ribonucleotides at every position except the 3′-terminus, whichcomprises two thymidine nucleotide overhangs.

FIG. 9 compares a siNA construct synthesized by the method of theinvention described in Example 1, wherein an inverted deoxyabasicsuccinate linker was used to generate a siNA having a 3′-inverteddeoxyabasic cap on the antisense strand of the siNA. This constructshows improved activity compared to the control siNA constructconsisting of all ribonucleotides at every position except the3′-terminus which comprises two thymidine nucleotide overhangs.

FIG. 10 shows the results of an RNAi activity screen of chemicallymodified siNA constructs including 3′-glyceryl modified siNA constructscompared to an all RNA control siNA construct using a luciferasereporter system. These chemically modified siNAs were compared in theluciferase assay described herein at 1 nM and 10 nM concentration usingan all RNA siNA control (siGL2) having 3′-terminal dithymidine (TT) andits corresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I. As shown in the Figure, the 3′-terminal modified siNAconstructs retain significant RNAi activity compared to the unmodifiedcontrol siNA (siGL2) construct.

FIG. 11 shows the results of an RNAi activity screen of chemicallymodified siNA constructs. The screen compared various combinations ofsense strand chemical modifications and antisense strand chemicalmodifications. These chemically modified siNAs were compared in theluciferase assay described herein at 1 nM and 10 nM concentration usingan all RNA siNA control (siGL2) having 3′-terminal dithymidine (TT) andits corresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I. As shown in the figure, the chemically modified Sirna/RPI30063/30430, Sirna/RPI 30433/30430, and Sirna/RPI 30063/30224 constructsretain significant RNAi activity compared to the unmodified control siNAconstruct. It should be noted that Sirna/RPI 30433/30430 is a siNAconstruct having no ribonucleotides which retains significant RNAiactivity compared to the unmodified control siGL2 construct in vitro,therefore, this construct is expected to have both similar RNAi activityand improved stability in vivo compared to siNA constructs havingribonucleotides.

FIG. 12 shows the results of an RNAi activity screen of chemicallymodified siNA constructs. The screen compared various combinations ofsense strand chemical modifications and antisense strand chemicalmodifications. These chemically modified siNAs were compared in theluciferase assay described herein at 1 nM and 10 nM concentration usingan all RNA siNA control (siGL2) having 3′-terminal dithymidine (TT) andits corresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I. As shown in the figure, the chemically modified Sirna/RPI30063/30224 and Sirna/RPI 30063/30430 constructs retain significant RNAiactivity compared to the control siNA (siGL2) construct. In addition,the antisense strand alone (Sirna/RPI 30430) and an inverted control(Sirna/RPI 30227/30229), having matched chemistry to Sirna/RPI(30063/30224) were compared to the siNA duplexes described above. Theantisense strand (Sirna/RPI 30430) alone provides far less inhibitioncompared to the siNA duplexes using this sequence.

FIG. 13 shows the results of an RNAi activity screen of chemicallymodified siNA constructs. The screen compared various combinations ofsense strand chemical modifications and antisense strand chemicalmodifications. These chemically modified siNAs were compared in theluciferase assay described herein at 1 nM and 10 nM concentration usingan all RNA siNA control (siGL2) having 3′-terminal dithymidine (TT) andits corresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I. In addition, an inverted control (Sirna/RPI 30226/30229, havingmatched chemistry to Sirna/RPI 30222/30224) was compared to the siNAduplexes described above. As shown in the figure, the chemicallymodified Sirna/RPI 28251/30430, Sirna/RPI 28251/30224, and Sirna/RPI30222/30224 constructs retain significant RNAi activity compared to thecontrol siNA construct, and the chemically modified Sirna/RPI28251/30430 construct demonstrates improved activity compared to thecontrol siNA (siGL2) construct.

FIG. 14 shows the results of an RNAi activity screen of chemicallymodified siNA constructs including various 3′-terminal modified siNAconstructs compared to an all RNA control siNA construct using aluciferase reporter system. These chemically modified siNAs werecompared in the luciferase assay described herein at 1 nM and 10 nMconcentration using an all RNA siNA control (siGL2) having 3′-terminaldithymidine (TT) and its corresponding inverted control (Inv siGL2). Thebackground level of luciferase expression in the HeLa cells isdesignated by the “cells” column. Sense and antisense strands ofchemically modified siNA constructs are shown by Sirna/RPI number (sensestrand/antisense strand). Sequences corresponding to these Sirna/RPInumbers are shown in Table I. As shown in the figure, the chemicallymodified Sirna/RPI 30222/30546, 30222/30224, 30222/30551, 30222/30557and 30222/30558 constructs retain significant RNAi activity compared tothe control siNA construct.

FIG. 15 shows the results of an RNAi activity screen of chemicallymodified siNA constructs. The screen compared various combinations ofsense strand chemistries compared to a fixed antisense strand chemistry.These chemically modified siNAs were compared in the luciferase assaydescribed herein at 1 nM and 10 nM concentration using an all RNA siNAcontrol (siGL2) having 3′-terminal dithymidine (TT) and itscorresponding inverted control (Inv siGL2). The background level ofluciferase expression in the HeLa cells is designated by the “cells”column. Sense and antisense strands of chemically modified siNAconstructs are shown by Sirna/RPI number (sense strand/antisensestrand). Sequences corresponding to these Sirna/RPI numbers are shown inTable I. As shown in the figure, the chemically modified Sirna/RPI30063/30430, 30434/30430, and 30435/30430 constructs all demonstrategreater activity compared to the control siNA (siGL2) construct.

Example 6: RNAi Activity Titration

A titration assay was performed to determine the lower range of siNAconcentration required for RNAi activity both in a control siNAconstruct consisting of all RNA nucleotides containing two thymidinenucleotide overhangs and a chemically modified siNA construct comprisingfive phosphorothioate internucleotide linkages in both the sense andantisense strands. The assay was performed as described above, however,the siNA constructs were diluted to final concentrations between 2.5 nMand 0.025 nM. Results are shown in FIG. 16. As shown in FIG. 16, thechemically modified siNA construct shows a very similar concentrationdependent RNAi activity profile to the control siNA construct whencompared to an inverted siNA sequence control.

Example 7: siNA Design

siNA target sites were chosen by analyzing sequences of the target RNAand optionally prioritizing the target sites on the basis of folding(structure of any given sequence analyzed to determine siNAaccessibility to the target), by using a library of siNA molecules asdescribed in Example 4, or alternately by using an in vitro siNA systemas described in Example 9 herein. siNA molecules were designed thatcould bind each target and are optionally individually analyzed bycomputer folding to assess whether the siNA molecule can interact withthe target sequence. Varying the length of the siNA molecules can bechosen to optimize activity. Generally, a sufficient number ofcomplementary nucleotide bases are chosen to bind to, or otherwiseinteract with, the target RNA, but the degree of complementarity can bemodulated to accommodate siNA duplexes or varying length or basecomposition. By using such methodologies, siNA molecules can be designedto target sites within any known RNA sequence, for example those RNAsequences corresponding to the any gene transcript.

Chemically modified siNA constructs are designed to provide nucleasestability for systemic administration in vivo and/or improvedpharmacokinetic, localization, and delivery properties while preservingthe ability to mediate RNAi activity. Chemical modifications asdescribed herein are introduced synthetically using synthetic methodsdescribed herein and those generally known in the art. The syntheticsiNA constructs are then assayed for nuclease stability in serum and/orcellular/tissue extracts (e.g. liver extracts). The synthetic siNAconstructs are also tested in parallel for RNAi activity using anappropriate assay, such as a luciferase reporter assay as describedherein or another suitable assay that can quantity RNAi activity.Synthetic siNA constructs that possess both nuclease stability and RNAiactivity can be further modified and re-evaluated in stability andactivity assays. The chemical modifications of the stabilized activesiNA constructs can then be applied to any siNA sequence targeting anychosen RNA and used, for example, in target screening assays to picklead siNA compounds for therapeutic development (see for example FIG.27).

Example 8: Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwise fashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphosphoramidite groups, and exocyclicamine protecting groups (e.g. N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2′-O-Silyl Ethers can be usedin conjunction with acid-labile 2′-O-orthoester protecting groups in thesynthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′-direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and Fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Deprotection and purification of the siNA can beperformed as is generally described in Usman et al., U.S. Pat. Nos.5,831,071, 6,353,098, 6,437,117, and Bellon et al., U.S. Pat. Nos.6,054,576, 6,162,909, 6,303,773, or Scaringe supra, incorporated byreference herein in their entireties. Additionally, deprotectionconditions can be modified to provide the best possible yield and purityof siNA constructs. For example, applicant has observed thatoligonucleotides comprising 2′-deoxy-2′-fluoro nucleotides can degradeunder inappropriate deprotection conditions. Such oligonucleotides aredeprotected using aqueous methylamine at about 35° C. for 30 minutes. Ifthe 2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 9: RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell free system is usedto evaluate siNA constructs specific to target RNA. The assay comprisesthe system described by Tuschl et al., 1999, Genes and Development, 13,3191-3197 and Zamore et al., 2000, Cell, 101, 25-33 adapted for use withtarget RNA. A Drosophila extract derived from syncytial blastoderm isused to reconstitute RNAi activity in vitro. Target RNA is generated viain vitro transcription from an appropriate plasmid using T7 RNApolymerase or via chemical synthesis as described herein. Sense andantisense siNA strands (for example 20 uM each) are annealed byincubation in buffer (such as 100 mM potassium acetate, 30 mM HEPES-KOH,pH 7.4, 2 mM magnesium acetate) for 1 minute at 90° C. followed by 1hour at 37° C., then diluted in lysis buffer (for example 100 mMpotassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate).Annealing can be monitored by gel electrophoresis on an agarose gel inTBE buffer and stained with ethidium bromide. The Drosophila lysate isprepared using zero to two-hour-old embryos from Oregon R fliescollected on yeasted molasses agar that are dechorionated and lysed. Thelysate is centrifuged and the supernatant isolated. The assay comprisesa reaction mixture containing 50% lysate [vol/vol], RNA (10-50 pM finalconcentration), and 10% [vol/vol] lysis buffer containing siNA (10 nMfinal concentration). The reaction mixture also contains 10 mM creatinephosphate, 10 ug·ml creatine phosphokinase, 100 um GTP, 100 uM UTP, 100uM CTP, 500 uM ATP, 5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM ofeach amino acid. The final concentration of potassium acetate isadjusted to 100 mM. The reactions are pre-assembled on ice andpreincubated at 25° C. for 10 minutes before adding RNA, then incubatedat 25° C. for an additional 60 minutes. Reactions are quenched with 4volumes of 1.25× Passive Lysis Buffer (Promega). Target RNA cleavage isassayed by RT-PCR analysis or other methods known in the art and arecompared to control reactions in which siNA is omitted from thereaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²p] CTP, passed over aG 50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by Phosphor Imager® quantitation ofbands representing intact control RNA or RNA from control reactionswithout siNA and the cleavage products generated by the assay.

In one embodiment, this assay is used to determine target sites the RNAtarget for siNA mediated RNAi cleavage, wherein a plurality of siNAconstructs are screened for RNAi mediated cleavage of the RNA target,for example, by analyzing the assay reaction by electrophoresis oflabeled target RNA, or by northern blotting, as well as by othermethodology well known in the art.

Example 10: Nucleic Acid Inhibition of Target RNA In Vivo

siNA molecules targeted to the target RNA are designed and synthesizedas described above. These nucleic acid molecules can be tested forcleavage activity in vivo, for example, using the following procedure.

Two formats are used to test the efficacy of siNAs targeting aparticular gene transcript. First, the reagents are tested on targetexpressing cells (e.g., HeLa), to determine the extent of RNA andprotein inhibition. siNA reagents are selected against the RNA target.RNA inhibition is measured after delivery of these reagents by asuitable transfection agent to cells. Relative amounts of target RNA aremeasured versus actin using real-time PCR monitoring of amplification(eg., ABI 7700 Taqman®). A comparison is made to a mixture ofoligonucleotide sequences made to unrelated targets or to a randomizedsiNA control with the same overall length and chemistry, but withrandomly substituted nucleotides at each position. Primary and secondarylead reagents are chosen for the target and optimization performed.After an optimal transfection agent concentration is chosen, a RNAtime-course of inhibition is performed with the lead siNA molecule. Inaddition, a cell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., HeLa) are seeded, for example, at 1×10⁵ cells per well of asix-well dish in EGM-2 (BioWhittaker) the day before transfection. siNA(final concentration, for example 20 nM) and cationic lipid (e.g., finalconcentration 2 g/ml) are complexed in EGM basal media (Biowhittaker) at37° C. for 30 mins in polystyrene tubes. Following vortexing, thecomplexed siNA is added to each well and incubated for the timesindicated. For initial optimization experiments, cells are seeded, forexample, at 1×10³ in 96 well plates and siNA complex added as described.Efficiency of delivery of siNA to cells is determined using afluorescent siNA complexed with lipid. Cells in 6-well dishes areincubated with siNA for 24 hours, rinsed with PBS and fixed in 2%paraformaldehyde for 15 minutes at room temperature. Uptake of siNA isvisualized using a fluorescent microscope.

Taqman and Lightcycler Quantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For Taqman analysis, dual-labeled probes aresynthesized with the reporter dye, FAM or JOE, covalently linked at the5′-end and the quencher dye TAMRA conjugated to the 3′-end. One-stepRT-PCR amplifications are performed on, for example, an ABI PRISM 7700Sequence Detector using 50 μl reactions consisting of 10 μl total RNA,100 nM forward primer, 900 nM reverse primer, 100 nM probe, 1× TaqManPCR reaction buffer (PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM eachdATP, dCTP, dGTP, and dTTP, 10 U RNase Inhibitor (Promega), 1.25 UAmpliTaq Gold (PE-Applied Biosystems) and 10 U M-MLV ReverseTranscriptase (Promega). The thermal cycling conditions can consist of30 min at 48° C., 10 min at 95° C., followed by 40 cycles of 15 sec at95° C. and 1 min at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/r×n) and normalizing to 1-actin or GAPDH mRNA inparallel TaqMan reactions. For each gene of interest an upper and lowerprimer and a fluorescently labeled probe are designed. Real timeincorporation of SYBR Green I dye into a specific PCR product can bemeasured in glass capillary tubes using a lightcyler. A standard curveis generated for each primer pair using control cRNA. Values arerepresented as relative expression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 11: Animal Models

Various animal models can be used to screen siNA constructs in vivo asare known in the art, for example those animal models that are used toevaluate other nucleic acid technologies such as enzymatic nucleic acidmolecules (ribozymes) and/or antisense. Such animal models are used totest the efficacy of siNA molecules described herein. In a non-limitingexample, siNA molecules that are designed as anti-angiogenic agents canbe screened using animal models. There are several animal modelsavailable in which to test the anti-angiogenesis effect of nucleic acidsof the present invention, such as siNA, directed against genesassociated with angiogenesis and/or metastais, such as VEGFR (e.g.,VEGFR1, VEGFR2, and VEGFR3) genes. Typically a corneal model has beenused to study angiogenesis in rat and rabbit, since recruitment ofvessels can easily be followed in this normally avascular tissue (Pandeyet al., 1995 Science 268: 567-569). In these models, a small Teflon orHydron disk pretreated with an angiogenesis factor (e.g. bFGF or VEGF)is inserted into a pocket surgically created in the cornea. Angiogenesisis monitored 3 to 5 days later. siNA molecules directed against VEGFRmRNAs would be delivered in the disk as well, or dropwise to the eyeover the time course of the experiment. In another eye model, hypoxiahas been shown to cause both increased expression of VEGF andneovascularization in the retina (Pierce et al., 1995 Proc. Natl. Acad.Sci. USA. 92: 905-909; Shweiki et al., 1992 J. Clin. Invest. 91:2235-2243).

Several animal models exist for screening of anti-angiogenic agents.These include corneal vessel formation following corneal injury (Burgeret al., 1985 Cornea 4: 35-41; Lepri, et al., 1994 J. Ocular Pharmacol.10: 273-280; Ormerod et al., 1990 Am. J. Pathol. 137: 1243-1252) orintracorneal growth factor implant (Grant et al., 1993 Diabetologia 36:282-291; Pandey et al. 1995 supra; Zieche et al., 1992 Lab. Invest. 67:711-715), vessel growth into Matrigel matrix containing growth factors(Passaniti et al., 1992 supra), female reproductive organneovascularization following hormonal manipulation (Shweiki et al., 1993Clin. Invest. 91: 2235-2243), several models involving inhibition oftumor growth in highly vascularized solid tumors (O'Reilly et al., 1994Cell 79: 315-328; Senger et al., 1993 Cancer and Metas. Rev. 12:303-324; Takahasi et al., 1994 Cancer Res. 54: 4233-4237; Kim et al.,1993 supra), and transient hypoxia-induced neovascularization in themouse retina (Pierce et al., 1995 Proc. Natl. Acad. Sci. USA. 92:905-909).gene

The cornea model, described in Pandey et al. supra, is the most commonand well characterized anti-angiogenic agent efficacy screening model.This model involves an avascular tissue into which vessels are recruitedby a stimulating agent (growth factor, thermal or alkali burn,endotoxin). The corneal model utilizes the intrastromal cornealimplantation of a Teflon pellet soaked in a VEGF-Hydron solution torecruit blood vessels toward the pellet, which can be quantitated usingstandard microscopic and image analysis techniques. To evaluate theiranti-angiogenic efficacy, siNA molecules are applied topically to theeye or bound within Hydron on the Teflon pellet itself. This avascularcornea as well as the Matrigel model (described below) provide for lowbackground assays. While the corneal model has been performedextensively in the rabbit, studies in the rat have also been conducted.

The mouse model (Passaniti et al., supra) is a non-tissue model whichutilizes Matrigel, an extract of basement membrane (Kleinman et al.,1986) or Millipore® filter disk, which can be impregnated with growthfactors and anti-angiogenic agents in a liquid form prior to injection.Upon subcutaneous administration at body temperature, the Matrigel orMillipore® filter disk forms a solid implant. VEGF embedded in theMatrigel or Millipore® filter disk is used to recruit vessels within thematrix of the Matrigel or Millipore® filter disk which can be processedhistologically for endothelial cell specific vWF (factor VIII antigen)immunohistochemistry, Trichrome-Masson stain, or hemoglobin content.Like the cornea, the Matrigel or Millipore® filter disk are avascular;however, it is not tissue. In the Matrigel or Millipore® filter diskmodel, siNA molecules are administered within the matrix of the Matrigelor Millipore® filter disk to test their anti-angiogenic efficacy. Thus,delivery issues in this model, as with delivery of siNA molecules byHydron-coated Teflon pellets in the rat cornea model, may be lessproblematic due to the homogeneous presence of the siNA within therespective matrix.

The Lewis lung carcinoma and B-16 murine melanoma models are wellaccepted models of primary and metastatic cancer and are used forinitial screening of anti-cancer agents. These murine models are notdependent upon the use of immunodeficient mice, are relativelyinexpensive, and minimize housing concerns. Both the Lewis lung and B-16melanoma models involve subcutaneous implantation of approximately 10⁶tumor cells from metastatically aggressive tumor cell lines (Lewis lunglines 3LL or D122, LLc-LN7; B-16-BL6 melanoma) in C57BL/6J mice.Alternatively, the Lewis lung model can be produced by the surgicalimplantation of tumor spheres (approximately 0.8 mm in diameter).Metastasis also may be modeled by injecting the tumor cells directlyintraveneously. In the Lewis lung model, microscopic metastases can beobserved approximately 14 days following implantation with quantifiablemacroscopic metastatic tumors developing within 21-25 days. The B-16melanoma exhibits a similar time course with tumor neovascularizationbeginning 4 days following implantation. Since both primary andmetastatic tumors exist in these models after 21-25 days in the sameanimal, multiple measurements can be taken as indices of efficacy.Primary tumor volume and growth latency as well as the number of micro-and macroscopic metastatic lung foci or number of animals exhibitingmetastases can be quantitated. The percent increase in lifespan can alsobe measured. Thus, these models would provide suitable primary efficacyassays for screening systemically administered siNA molecules and siNAformulations.

In the Lewis lung and B-16 melanoma models, systemic pharmacotherapywith a wide variety of agents usually begins 1-7 days following tumorimplantation/inoculation with either continuous or multipleadministration regimens. Concurrent pharmacokinetic studies can beperformed to determine whether sufficient tissue levels of siNA can beachieved for pharmacodynamic effect to be expected. Furthermore, primarytumors and secondary lung metastases can be removed and subjected to avariety of in vitro studies (i.e. target RNA reduction).

Ohno-Matsui et al., 2002, Am. J. Pathology, 160, 711-719 describe amodel of severe proliferative retinopathy and retinal detachment in miceunder inducible expression of vascular endothelial growth factor. Inthis model, expression of a VEGF transgene results in elevated levels ofocular VEGF that is associated with severe proliferative retinopathy andretinal detachment. Furthermore, Mori et al., 2001, J. CellularPhysiology, 188, 253-263, describe a model of laser induced choroidalneovascularization that can be used in conjunction with intravitreous orsubretianl injection of siNA molecules of the invention to evaluate theefficacy of siNA treatment of severe proliferative retinopathy andretinal detachment.

In utilizing these models to assess siNA activity, VEGFR1, VEGFR2,and/or VEGFR3 protein levels can be measured clinically orexperimentally by FACS analysis. VEGFR1, VEGFR2, and/or VEGFR3 encodedmRNA levels can be assessed by Northern analysis, RNase-protection,primer extension analysis and/or quantitative RT-PCR. siNA moleculesthat block VEGFR1, VEGFR2, and/or VEGFR3 protein encoding mRNAs andtherefore result in decreased levels of VEGFR1, VEGFR2, and/or VEGFR3activity by more than 20% in vitro can be identified using thetechniques described herein.

Example 12: siNA-Mediated Inhibition of Angiogenesis In Vivo

The purpose of this study was to assess the anti-angiogenic activity ofsiNA targeted against VEGFR1, using the rat cornea model of VEGF inducedangiogenesis discussed in Example 11 above). The siNA molecules shown inFIG. 23 have matched inverted controls which are inactive since they arenot able to interact with the RNA target. The siNA molecules and VEGFwere co-delivered using the filter disk method. Nitrocellulose filterdisks (Millipore®) of 0.057 diameter were immersed in appropriatesolutions and were surgically implanted in rat cornea as described byPandey et al., supra.

The stimulus for angiogenesis in this study was the treatment of thefilter disk with 30 μM VEGF which is implanted within the cornea'sstroma. This dose yields reproducible neovascularization stemming fromthe pericorneal vascular plexus growing toward the disk in adose-response study 5 days following implant. Filter disks treated onlywith the vehicle for VEGF show no angiogenic response. The siNA wereco-adminstered with VEGF on a disk in three different siNAconcentrations. One concern with the simultaneous administration is thatthe siNA would not be able to inhibit angiogenesis since VEGF receptorscan be stimulated. However, Applicant has observed that in low VEGFdoses, the neovascular response reverts to normal suggesting that theVEGF stimulus is essential for maintaining the angiogenic response.Blocking the production of VEGF receptors using simultaneousadministration of anti-VEGF-R mRNA siNA could attenuate the normalneovascularization induced by the filter disk treated with VEGF.

Materials and Methods:

Test Compounds and Controls

-   -   R&D Systems VEGF, carrier free at 75 μM in 82 mM Tris-C1, pH 6.9    -   siNA, 1.67 μG/μL, SITE 2340 (SIRNA/RPI 29695/29699)        sense/antisense    -   siNA, 1.67 μG/μL, INVERTED CONTROL FOR SITE 2340 (SIRNA/RPI        29983/29984) sense/antisense    -   siNA 1.67 μg/μL, Site 2340 (Sirna/RPI 30196/30416)        sense/antisense        Animals    -   Harlan Sprague-Dawley Rats, Approximately 225-250 g    -   45 males, 5 animals per group.        Husbandry

Animals are housed in groups of two. Feed, water, temperature andhumidity are determined according to Pharmacology Testing Facilityperformance standards (SOP's) which are in accordance with the 1996Guide for the Care and Use of Laboratory Animals (NRC). Animals areacclimated to the facility for at least 7 days prior to experimentation.During this time, animals are observed for overall health and sentinelsare bled for baseline serology.

Experimental Groups

Each solution (VEGF and siNAs) was prepared as a 1× solution for finalconcentrations shown in the experimental groups described in Table III.

siNA Annealing Conditions

siNA sense and antisense strands are annealed for 1 minute in H₂O at1.67 mg/mL/strand followed by a 1 hour incubation at 37° C. producing3.34 mg/mL of duplexed siNA. For the 20 μg/eye treatment, 6 μLs of the3.34 mg/mL duplex is injected into the eye (see below). The 3.34 mg/mLduplex siNA can then be serially diluted for dose response assays.

Preparation of VEGF Filter Disk

For corneal implantation, 0.57 mm diameter nitrocellulose disks,prepared from 0.45 μm pore diameter nitrocellulose filter membranes(Millipore Corporation), were soaked for 30 min in 1 μL of 75 μM VEGF in82 mM Tris-HCl (pH 6.9) in covered petri dishes on ice. Filter diskssoaked only with the vehicle for VEGF (83 mM Tris-C1 pH 6.9) elicit noangiogenic response.

Corneal Surgery

The rat corneal model used in this study was a modified from Koch et al.Supra and Pandey et al., supra. Briefly, corneas were irrigated with0.5% povidone iodine solution followed by normal saline and two drops of2% lidocaine. Under a dissecting microscope (Leica MZ-6), a stromalpocket was created and a presoaked filter disk (see above) was insertedinto the pocket such that its edge was 1 mm from the corneal limbus.

Intraconjunctival Injection of Test Solutions

Immediately after disk insertion, the tip of a 40-50 μm OD injector(constructed in our laboratory) was inserted within the conjunctivaltissue 1 mm away from the edge of the corneal limbus that was directlyadjacent to the VEGF-soaked filter disk. Six hundred nanoliters of testsolution (siNA, inverted control or sterile water vehicle) weredispensed at a rate of 1.2 L/min using a syringe pump (Kd Scientific).The injector was then removed, serially rinsed in 70% ethanol andsterile water and immersed in sterile water between each injection. Oncethe test solution was injected, closure of the eyelid was maintainedusing microaneurism clips until the animal began to recover gross motoractivity. Following treatment, animals were warmed on a heating pad at37° C.

Quantitation of Angiogenic Response

Five days after disk implantation, animals were euthanized followingadministration of 0.4 mg/kg atropine and corneas were digitally imaged.The neovascular surface area (NSA, expressed in pixels) was measuredpostmortem from blood-filled corneal vessels using computerizedmorphometry (Image Pro Plus, Media Cybernetics, v2.0). The individualmean NSA was determined in triplicate from three regions of identicalsize in the area of maximal neovascularization between the filter diskand the limbus. The number of pixels corresponding to the blood-filledcorneal vessels in these regions was summated to produce an index ofNSA. A group mean NSA was then calculated. Data from each treatmentgroup were normalized to VEGF/siNA vehicle-treated control NSA andfinally expressed as percent inhibition of VEGF-induced angiogenesis.

Statistics

After determining the normality of treatment group means, group meanpercent inhibition of VEGF-induced angiogenesis was subjected to aone-way analysis of variance. This was followed by two post-hoc testsfor significance including Dunnett's (comparison to VEGF control) andTukey-Kramer (all other group mean comparisons) at alpha=0.05.Statistical analyses were performed using JMP v.3.1.6 (SAS Institute).

Results of the study are graphically represented in FIGS. 23 and 76. Asshown in FIG. 23, VEGFr1 site 4229 active siNA (Sirna/RPI 29695/29699)at three concentrations were effective at inhibiting angiogenesiscompared to the inverted siNA control (Sirna/RPI 29983/29984) and theVEGF control. A chemically modified version of the VEGFr1 site 4229active siNA comprising a sense strand having 2′-deoxy-2′-fluoropyrimidines and ribo purines with 5′ and 3′ terminal inverteddeoxyabasic residues and an antisense strand having 2′-deoxy-2′-fluoropyrimidines and ribo purines with a terminal 3′-phosphorothioateinternucleotide linkage (Sirna/RPI 30196/30416), showed similarinhibition. Furthermore, VEGFr1 site 349 active siNA having “Stab 9/10”chemistry (Sirna #31270/31273) was tested for inhibition of VEGF-inducedangiogenesis at three different concentrations (2.0 ug, 1.0 ug, and 0.1μg dose response) as compared to a matched chemistry inverted controlsiNA construct (Sirna #31276/31279) at each concentration and a VEGFcontrol in which no siNA was administered. As shown in FIG. 76, theactive siNA construct having “Stab 9/10” chemistry (Sirna #31270/31273)is highly effective in inhibiting VEGF-induced angiogenesis in the ratcorneal model compared to the matched chemistry inverted control siNA atconcentrations from 0.1 μg to 2.0 ug. These results demonstrate thatsiNA molecules having different chemically modified compositions, suchas the modifications described herein, are capable of significantlyinhibiting angiogenesis in vivo.

Example 13: Inhibition of HBV Using siNA Molecules of the Invention

Transfection of HepG2 Cells with psHBV-1 and siNA

The human hepatocellular carcinoma cell line Hep G2 was grown inDulbecco's modified Eagle media supplemented with 10% fetal calf serum,2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate,25 mM Hepes, 100 units penicillin, and 100 μg/ml streptomycin. Togenerate a replication competent cDNA, prior to transfection the HBVgenomic sequences are excised from the bacterial plasmid sequencecontained in the psHBV-1 vector. Other methods known in the art can beused to generate a replication competent cDNA. This was done with anEcoRI and Hind III restriction digest. Following completion of thedigest, a ligation was performed under dilute conditions (20 μg/ml) tofavor intermolecular ligation. The total ligation mixture was thenconcentrated using Qiagen spin columns.

siNA Activity Screen and Dose Response Assay

Transfection of the human hepatocellular carcinoma cell line, Hep G2,with replication-competent HBV DNA results in the expression of HBVproteins and the production of virions. To test the efficacy of siNAstargeted against HBV RNA, several siNA duplexes targeting differentsites within HBV pregenomic RNA were co-transfected with HBV genomic DNAonce at 25 nM with lipid at 12.5 ug/ml into Hep G2 cells, and thesubsequent levels of secreted HBV surface antigen (HBsAg) were analyzedby ELISA (see FIG. 24). Inverted sequence duplexes were used as negativecontrols. Subsequently, dose response studies were performed in whichthe siNA duplexes were co-transfected with HBV genomic DNA at 0.5, 5, 10and 25 nM with lipid at 12.5 ug/ml into Hep G2 cells, and the subsequentlevels of secreted HBV surface antigen (HBsAg) were analyzed by ELISA(see FIG. 25).

Analysis of HBsAg Levels Following siNA Treatment

To determine siNA activity, HbsAg levels were measured followingtransfection with siNA. Immulon 4 (Dynax) microtiter wells were coatedovernight at 4° C. with anti-HBsAg Mab (Biostride B88-95-31ad,ay) at 1gg/ml in Carbonate Buffer (Na2CO3 15 mM, NaHCO₃35 mM, pH 9.5). The wellswere then washed 4× with PBST (PBS, 0.05% Tween® 20) and blocked for 1hr at 37° C. with PBST, 1% BSA. Following washing as above, the wellswere dried at 37° C. for 30 min. Biotinylated goat ant-HBsAg (AccurateYVS1807) was diluted 1:1000 in PBST and incubated in the wells for 1 hr.at 37° C. The wells were washed 4× with PBST. Streptavidin/AlkalinePhosphatase Conjugate (Pierce 21324) was diluted to 250 ng/ml in PBST,and incubated in the wells for 1 hr. at 37° C. After washing as above,p-nitrophenyl phosphate substrate (Pierce 37620) was added to the wells,which were then incubated for 1 hour at 37° C. The optical density at405 nm was then determined. Results of the HBV screen study aresummarized in FIG. 24, whereas the results of a dose response assayusing lead siNA constructs targeting sites 262 and 1580 of the HBVpregenomic RNA are shown in FIG. 25. As shown in FIG. 25, the siNAconstructs targeting sites 262 and 1580 of HBV RNA provides significantdose response inhibition of viral replication/activity when compared toinverted siNA controls.

Comparison of Different Chemically Stabilized siNA Motifs Targeting HBVRNA Site 1580

Two different siNA stabilization chemistries were compared in a doseresponse HBsAg assay using inverted matched chemistry controls. The“Stab7/8” (Table IV) constructs comprise a sense strand having2′-deoxy-2′-fluoro pyrimidine nucleotides and 2′-deoxy purinenucleotides with 5′ and 3′ terminal inverted deoxyabasic residues and anantisense strand having 2′-deoxy-2′-fluoro pyrimidine nucleotides and2′-O-methyl purine nucleotides with a terminal 3′ phosphorothioatelinkage. The “Stab7/11 (Table IV) constructs comprise a sense strandhaving 2′-deoxy-2′-fluoro pyrimidine nucleotides and 2′-deoxy purinenucleotides with 5′ and 3′ terminal inverted deoxyabasic residues and anantisense strand having 2′-deoxy-2′-fluoro pyrimidine nucleotides and2′-deoxy purine nucleotides with a terminal 3′ phosphorothioate linkage(see for example Table I). As shown in FIG. 26, the chemicallystabilized siNA constructs both show significant inhibition of HBVantigen in a dose dependent manner compared to matched invertedcontrols. A separate direct screen of Stab 7/8 constructs targeting HBVRNAin HepG2 cells that identified stabilized siNA constructs with potentactivity is shown in FIG. 87.

Time Course Evaluation of Different Chemically Stabilized siNA MotifsTargeting HBV RNA Site 1580

Four different siNA constructs having different stabilizationchemistries were compared to an unstabilized siRNA construct in a doseresponse time course HBsAg assay, the results of which are shown inFIGS. 28-31. The different constructs were compared to an unstabilizedribonucleotide control siRNA construct (Sirna/RPI #30287/30298) atdifferent concentrations (5 nM, 10 nM, 25 nM, 50 nM, and 100 nM) overthe course of nine days. Activity based on HBsAg levels was determinedat day 3, day 6, and day 9. The “Stab 4/5” (Table IV) constructscomprise a sense strand (Sirna/RPI #30355) having 2′-deoxy-2′-fluoropyrimidine nucleotides and purine ribonucleotides with 5′ and 3′terminal inverted deoxyabasic residues and an antisense strand(Sirna/RPI #30366) having 2′-deoxy-2′-fluoro pyrimidine nucleotides andpurine ribonucleotides with a terminal 3′ phosphorothioate linkage (datashown in FIG. 28). The “Stab7/8” (Table IV) constructs comprise a sensestrand (Sirna/RPI #30612) having 2′-deoxy-2′-fluoro pyrimidinenucleotides and 2′-deoxy purine nucleotides with 5′ and 3′ terminalinverted deoxyabasic residues and an antisense strand (Sirna/RPI #30620)having 2′-deoxy-2′-fluoro pyrimidine nucleotides and 2′-O-methyl purinenucleotides with a terminal 3′ phosphorothioate linkage (data shown inFIG. 29). The “Stab7/11 (Table IV) constructs comprise a sense(Sirna/RPI #30612) strand having 2′-deoxy-2′-fluoro pyrimidinenucleotides and 2′-deoxy purine nucleotides with 5′ and 3′ terminalinverted deoxyabasic residues and an antisense strand (Sirna/RPI #31175)having 2′-deoxy-2′-fluoro pyrimidine nucleotides and 2′-deoxy purinenucleotides with a terminal 3′ phosphorothioate linkage (data shown inFIG. 30). The “Stab9/10 (Table IV) constructs comprise a sense(Sirna/RPI #31335) strand having ribonucleotides with 5′ and 3′ terminalinverted deoxyabasic residues and an antisense strand (Sirna/RPI #31337)having ribonucleotides with a terminal 3′ phosphorothioate linkage (datashown in FIG. 31). As shown in FIGS. 28-31, the chemically stabilizedsiNA constructs all show significantly greater inhibition of HBV antigenin a dose dependent manner over the time course experiment compared tothe unstabilized siRNA construct.

A second study was performed using the stab 4/5 (Sirna 30355/30366),stab 7/8 (Sirna 30612/30620), and stab 7/11 (Sirna 30612/31175) siNAconstructs described above to examine the duration of effect of themodified siNA constructs out to 21 days post transfection compared to anall RNA control siNA (Sirna 30287/30298). A single transfection wasperformed with siRNAs targeted to HBV site 1580 and the culture mediawas subsequently replaced every three days. Secreted HBsAg levels weremonitored for at 3, 6, 9, 12, 15, 18 and 21 days post-transfection.FIGS. 77A-77F show activity of siNAs in reduction of HBsAg levelscompared to matched inverted controls at FIG. 77A. 3 days, FIG. 77B. 9days, and FIG. 77C. 21 days post transfection. Also shown is thecorresponding percent inhibition as function of time at siNAconcentrations of FIG. 77D. 100 nM, FIG. 77E. 50 nM, and FIG. 77F. 25nM.

Example 14: Inhibition of HCV Using siNA Molecules of the Invention

siNA Inhibition of a Chimeric HCV/Poliovirus in HeLa Cells

Inhibition of a chimeric HCV/Poliovirus was investigated using 21nucleotide siNA duplexes in HeLa cells. Seven siNA constructs weredesigned that target three regions in the highly conserved 5′untranslated region (UTR) of HCV RNA. The siNAs were screened in twocell culture systems dependent upon the 5′-UTR of HCV; one requirestranslation of an HCV/luciferase gene, while the other involvesreplication of a chimeric HCV/poliovirus (PV) (see Blatt et al., U.S.Ser. No. 09/740,332, filed Dec. 18, 2000, incorporated by referenceherein). Two siNAs (29579/29586; 29578/29585) targeting the same region(shifted by one nucleotide) are active in both systems (see FIG. 32) ascompared with inverse control siNA (29593/29600). For example, a >85%reduction in HCVPV replication was observed in siNA-treated cellscompared to an inverse siNA control (FIG. 32) with an IC50=−2.5 nM (FIG.33). To develop nuclease-resistant siNA for in vivo applications, siNAscan be modified to contain stabilizing chemical modifications. Suchmodifications include phosphorothioate linkages (P═S), 2′-O-methylnucleotides, 2′-fluoro (F) nucleotides, 2′-deoxy nucleotides, universalbase nucleotides, 5′ and/or 3′ end modifications and a variety of othernucleotide and non-nucleotide modifications, in one or both siNAstrands. Several of these constructs were tested in the HCV/polioviruschimera system, demonstrating significant reduction in viral replication(FIGS. 34-37). siNA constructs shown in FIGS. 34-37 are referred to bySirna/RPI #s that are cross referenced to Table III, which shows thesequence and chemical modifications of the constructs. siNA activity iscompared to relevant controls (untreated cells, scrambled/inactivecontrol sequences, or transfection controls). As shown in the Figures,siNA constructs of the invention provide potent inhibition of HCV RNA inthe HCV/poliovirus chimera system. As such, siNA constructs, includingchemically modified, nuclease resistant siNA molecules, represent animportant class of therapeutic agents for treating chronic HCVinfection.

siNA Inhibition of a HCV RNA Expression in a HCV Replicon System

In addition, a HCV replicon system was used to test the efficacy ofsiNAs targeting HCV RNA. The reagents are tested in cell culture usingHuh7 cells (see for example Randall et al., 2003, PNAS USA, 100,235-240) to determine the extent of RNA and protein inhibition. siNAwere selected against the HCV target as described herein. RNA inhibitionwas measured after delivery of these reagents by a suitable transfectionagent to Huh7 cells. Relative amounts of target RNA are measured versusactin using real-time PCR monitoring of amplification (eg., ABI 7700Taqman®). A comparison is made to a mixture of oligonucleotide sequencesdesigned to target unrelated targets or to a randomized siNA controlwith the same overall length and chemistry, but with randomlysubstituted nucleotides at each position. Primary and secondary leadreagents were chosen for the target and optimization performed. After anoptimal transfection agent concentration is chosen, a RNA time-course ofinhibition is performed with the lead siNA molecule. In addition, acell-plating format can be used to determine RNA inhibition. Anon-limiting example of a multiple target screen to assay siNA mediatedinhibition of HCV RNA is shown in FIG. 38. siNA reagents (Table I) weretransfected at 25 nM into Huh7 cells and HCV RNA quantitated compared tountreated cells (“cells” column in the figure) and cells transfectedwith lipofectamine (“LFA2K” column in the figure). As shown in theFigure, several siNA constructs show significant inhibition of HCV RNAexpression in the Huh7 replicon system. Chemically modified siNAconstructs were then screened as described above, with a non-limitingexample of a Stab 7/8 (see Table IV) chemistry siNA construct screenshown in FIG. 40. A follow up dose response study using chemicallymodified siNA constructs (Stab 4/5, see Table IV) at concentrations of 5nM, 10 nM, 25 nM and 100 nM compared to matched chemistry invertedcontrols is shown in FIG. 39, whereas a dose response study for Stab 7/8constructs at concentrations of 5 nM, 10 nM, 25 nM, 50 nM and 100 nMcompared to matched chemistry inverted controls is shown in FIG. 41. Aseparate direct screen of Stab 7/8 constructs targeting HCV RNA thatidentified stabilized siNA constructs with potent activity is shown inFIG. 86.

Example 15: Target Discovery in Mammalian Cells Using siNA Molecules

In a non-limiting example, compositions and methods of the invention areused to discover genes involved in a process of interest withinmammalian cells, such as cell growth, proliferation, apoptosis,morphology, angiogenesis, differentiation, migration, viralmultiplication, drug resistance, signal transduction, cell cycleregulation, or temperature sensitivity or other process. First, arandomized siNA library is generated.

These constructs are inserted into a vector capable of expressing a siNAfrom the library inside mammalian cells. Alternately, a pool ofsynthetic siNA molecules is generated.

Reporter System

In order to discover genes playing a role in the expression of certainproteins, such as proteins involved in a cellular process describedherein, a readily assayable reporter system is constructed in which areporter molecule is co-expressed when a particular protein of interestis expressed. The reporter system consists of a plasmid constructbearing a gene coding for a reporter gene, such as Green FluorescentProtein (GFP) or other reporter proteins known and readily available inthe art. The promoter region of the GFP gene is replaced by a portion ofa promoter for the protein of interest sufficient to direct efficienttranscription of the GFP gene. The plasmid can also contain a drugresistance gene, such as neomycin resistance, in order to select cellscontaining the plasmid.

Host Cell Lines for Target Discovery

A cell line is selected as host for target discovery. The cell line ispreferably known to express the protein of interest, such that upstreamgenes controlling the expression of the protein can be identified whenmodulated by a siNA construct expressed therein. The cells preferablyretain protein expression characteristics in culture. The reporterplasmid is transfected into cells, for example, using a cationic lipidformulation. Following transfection, the cells are subjected to limitingdilution cloning, for example, under selection by 600 μg/mL Geneticin.Cells retaining the plasmid survive the Geneticin treatment and formcolonies derived from single surviving cells. The resulting clonal celllines are screened by flow cytometry for the capacity to upregulate GFPproduction.

Treating the cells with, for example, sterilized M9 bacterial medium inwhich Pseudomonas aeruginosa had been cultured (Pseudomonas conditionedmedium, PCM) is used to induce the promoter. The PCM is supplementedwith phorbol myristate acetate (PMA). A clonal cell line highlyresponsive to promoter induction is selected as the reporter line forsubsequent studies.

siNA Library Construction

A siNA library was constructed with oligonucleotides containing hairpinsiNA constructs having randomized antisense regions and selfcomplementary sense regions. The library is generated synthesizing siNAconstructs having randomized sequence. Alternately, the siNA librariesare constructed as described in Usman et al., U.S. Ser. No. 60/402,996(incorporated by reference herein) Oligo sequence 5′ and 3′ of the siNAcontains restriction endonuclease cleavage sites for cloning. The 3′trailing sequence forms a stem-loop for priming DNA polymerase extensionto form a hairpin structure. The hairpin DNA construct is melted at 90°C. allowing DNA polymerase to generate a dsDNA construct. Thedouble-stranded siNA library is cloned into, for example, a U6+27transcription unit located in the 5′ LTR region of a retroviral vectorcontaining the human nerve growth factor receptor (hNGFr) reporter gene.Positioning the U6+27/siNA transcription unit in the 5′ LTR results in aduplication of the transcription unit when the vector integrates intothe host cell genome. As a result, the siNA is transcribed by RNApolymerase III from U6+27 and by RNA polymerase II activity directed bythe 5′ LTR. The siNA library is packaged into retroviral particles thatare used to infect and transduce clonal cells selected above. Assays ofthe hNGFr reporter are used to indicate the percentage of cells thatincorporated the siNA construct. By randomized region is meant a regionof completely random sequence and/or partially random sequence. Bycompletely random sequence is meant a sequence wherein theoreticallythere is equal representation of A, T, G and C nucleotides or modifiedderivatives thereof, at each position in the sequence. By partiallyrandom sequence is meant a sequence wherein there is an unequalrepresentation of A, T, G and C nucleotides or modified derivativesthereof, at each position in the sequence. A partially random sequencecan therefore have one or more positions of complete randomness and oneor more positions with defined nucleotides.

Enriching for Non-Responders to Induction

Sorting of siNA library-containing cells is performed to enrich forcells that produce less reporter GFP after treatment with the promoterinducers PCM and PMA. Lower GFP production cancan be due to RNAiactivity against genes involved in the activation of the mucin promoter.Alternatively, siNA can directly target the mucin/GFP transcriptresulting in reduced GFP expression.

Cells are seeded at a certain density, such as 1×10⁶ per 150 cm² stylecell culture flasks and grown in the appropriate cell culture mediumwith fetal bovine serum. After 72 hours, the cell culture medium isreplaced with serum-free medium. After 24 hours of serum deprivation,the cells are treated with serum-containing medium supplemented with PCM(to 40%) and PMA (to 50 nM) to induced GFP production. After 20 to 22hours, cells are monitored for GFP level on, for example, a FACStar Pluscell sorter. Sorting is performed if ≥90% of siNA library cells from anunsorted control sample were induced to produce GFP above backgroundlevels. Two cell fractions are collected in each round of sorting.Following the appropriate round of sorting, the M1 fraction is selectedto generate a database of siNA molecules present in the sorted cells.

Recovery of siNA Sequence from Sorted Cells

Genomic DNA is obtained from sorted siNA library cells by standardmethods. Nested polymerase chain reaction (PCR) primers that hybridizedto the retroviral vector 5′ and 3′ of the siNA are used to recover andamplify the siNA sequences from the particular clone of library cellDNA. The PCR product is ligated into a bacterial cloning vector. Therecovered siNA library in plasmid form can be used to generate adatabase of siNA sequences. For example, the library is cloned into E.coli. DNA is prepared by plasmid isolation from bacterial colonies or bydirect colony PCR and siNA sequence is determined. A second method canuse the siNA library to transfect cloned cells. Clonal lines of stablytransfected cells are established and induced with, for example, PCM andPMA. Those lines which fail to respond to GFP induction are probed byPCR for single siNA integration events. The unique siNA sequencesobtained by both methods are added to a Target Sequence Tag (TST)database.

Bioinformatics

The antisense region sequences of the isolated siNA constructs arecompared to public and private gene data banks. Gene matches arecompiled according to perfect and imperfect matches. Potential genetargets are categorized by the number of different siNA sequencesmatching each gene. Genes with more than one perfect siNA match areselected for Target Validation studies.

Validation of the Target Gene

To validate a target as a regulator of protein expression, siNA reagentsare designed to the target gene cDNA sequence from Genbank. The siNAreagents are complexed with a cationic lipid formulation prior toadministration to cloned cells at appropriate concentrations (e.g. 5-50nM or less). Cells are treated with siNA reagents, for example from 72to 96 hours. Before the termination of siNA treatment, PCM (to 40%) andPMA (to 50 nM), for example, are added to induce the promoter. Aftertwenty hours of induction the cells are harvested and assayed forphenotypic and molecular parameters. Reduced GFP expression in siNAtreated cells (measured by flow cytometry) is taken as evidence forvalidation of the target gene. Knockdown of target RNA in siNA treatedcells can correlate with reduced endogenous RNA and reduced GFP RNA tocomplete validation of the target.

Example 16: Screening siNA Constructs for Improved Pharmacokinetics

In a non-limiting example, siNA constructs are screened in vivo forimproved pharmacokinetic properties compared to all RNA or unmodifiedsiNA constructs. Chemical modifications are introduced into the siNAconstruct based on educated design parameters (e.g. introducing2′-mofications, base modifications, backbone modifications, terminal capmodifications, or covalently attached conjugates etc). The modifiedconstruct in tested in an appropriate system (e.g human serum fornuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, localized delivery, cellular uptake, and RNAiactivity.

Example 17: Indications

The siNA molecules of the invention can be used to treat a variety ofdiseases and conditions through modulation of gene expression. Using themethods described herein, chemically modified siNA molecules can bedesigned to modulate the expression any number of target genes,including but not limited to genes associated with cancer, metabolicdiseases, infectious diseases such as viral, bacterial or fungalinfections, neurologic diseases, musculoskeletal diseases, diseases ofthe immune system, diseases associated with signaling pathways andcellular messengers, and diseases associated with transport systemsincluding molecular pumps and channels.

Non-limiting examples of various viral genes that can be targeted usingsiNA molecules of the invention include Hepatitis C Virus (HCV, forexample Genbank Accession Nos: D11168, D50483.1, L38318 and S82227),Hepatitis B Virus (HBV, for example GenBank Accession No. AF100308.1),Human Immunodeficiency Virus type 1 (HIV-1, for example GenBankAccession No. U51188), Human Immunodeficiency Virus type 2 (HIV-2, forexample GenBank Accession No. X60667), West Nile Virus (WNV for exampleGenBank accession No. NC_001563), cytomegalovirus (CMV for exampleGenBank Accession No. NC_001347), respiratory syncytial virus (RSV forexample GenBank Accession No. NC_001781), influenza virus (for exampleGenBank Accession No. AF037412, rhinovirus (for example, GenBankaccession numbers: D00239, X02316, X01087, L24917, M16248, K02121,X01087), papillomavirus (for example GenBank Accession No. NC_001353),Herpes Simplex Virus (HSV for example GenBank Accession No. NC_001345),and other viruses such as HTLV (for example GenBank Accession No.AJ430458). Due to the high sequence variability of many viral genomes,selection of siNA molecules for broad therapeutic applications wouldlikely involve the conserved regions of the viral genome. Nonlimitingexamples of conserved regions of the viral genomes include but are notlimited to 5′-Non Coding Regions (NCR), 3′-Non Coding Regions (NCR) LTRregions and/or internal ribosome entry sites (IRES). siNA moleculesdesigned against conserved regions of various viral genomes will enableefficient inhibition of viral replication in diverse patient populationsand may ensure the effectiveness of the siNA molecules against viralquasi species which evolve due to mutations in the non-conserved regionsof the viral genome.

Non-limiting examples of human genes that can be targeted using siNAmolecules of the invention using methods described herein include anyhuman RNA sequence, for example those commonly referred to by GenbankAccession Number. These RNA sequences can be used to design siNAmolecules that inhibit gene expression and therefore abrogate diseases,conditions, or infections associated with expression of those genes.Such non-limiting examples of human genes that can be targeted usingsiNA molecules of the invention include VEGF (for example GenBankAccession No. NM_003376.3), VEGFr (VEGFR1 for example GenBank AccessionNo. XM_067723, VEGFR2 for example GenBank Accession No. AF063658), HER1,HER2, HER3, and HER4 (for example Genbank Accession Nos: NM_005228,NM_004448, NM_001982, and NM_005235 respectively), telomerase (TERT, forexample GenBank Accession No. NM_003219), telomerase RNA (for exampleGenBank Accession No. U86046), NFkappaB, Rel-A (for example GenBankAccession No. NM_005228), NOGO (for example GenBank Accession No.AB020693), NOGOr (for example GenBank Accession No. XM_015620), RAS (forexample GenBank Accession No. NM_004283), RAF (for example GenBankAccession No. XM_033884), CD20 (for example GenBank Accession No.X07203), METAP2 (for example GenBank Accession No. NM_003219), CLCA1(for example GenBank Accession No. NM_001285), phospholamban (forexample GenBank Accession No. NM_002667), PTP1B (for example GenBankAccession No. M31724), PCNA (for example GenBank Accession No.NM_002592.1), PKC-alpha (for example GenBank Accession No. NM_002737)and others. The genes described herein are provided as non-limitingexamples of genes that can be targeted using siNA molecules of theinvention. Additional examples of such genes are described by accessionnumber in Beigelman et al., U.S. Ser. No. 60/363,124, filed Mar. 11,2002 and incorporated by reference herein in its entirety.

The siNA molecule of the invention can also be used in a variety ofagricultural applications involving modulation of endogenous orexogenous gene expression in plants using siNA, including use asinsecticidal, antiviral and anti-fungal agents or modulate plant traitssuch as oil and starch profiles and stress resistance.

Example 18: Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with a siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

Example 19: Synthesis of siNA Conjugates

The introduction of conjugate moieties to siNA molecules of theinvention is accomplished either during solid phase synthesis usingphosphoramidite chemistry described above, or post-synthetically using,for example, N-hydroxysuccinimide (NHS) ester coupling to an aminolinker present in the siNA. Typically, a conjugate introduced duringsolid phase synthesis will be added to the 5′-end of a nucleic acidsequence as the final coupling reaction in the synthesis cycle using thephosphoramidite approach. Coupling conditions can be optimized for highyield coupling, for example by modification of coupling times andreagent concentrations to effectuate efficient coupling. As such, the5′-end of the sense strand of a siNA molecule is readily conjugated witha conjugate moiety having a reactive phosphorus group available forcoupling (e.g., a compound having Formulae 1, 5, 8, 55, 56, 57, 60, 86,92, 104, 110, 113, 115, 116, 117, 118, 120, or 122) using thephosphoramidite approach, providing a 5′-terminal conjugate (see forexample FIG. 65).

Conjugate precursors having a reactive phosphorus group and a protectedhydroxyl group can be used to incorporate a conjugate moiety anywhere inthe siNA sequence, such as in the loop portion of a single strandedhairpin siNA construct (see for example FIG. 66). For example, using thephosphoramidite approach, a conjugate moiety comprising aphosphoramidite and protected hydroxyl (e.g., a compound having Formulae86, 92, 104, 113, 115, 116, 117, 118, 120, or 122 herein) is firstcoupled at the desired position within the siNA sequence using solidphase synthesis phosphoramidite coupling. Second, removal of theprotecting group (e.g., dimethoxytrityl) allows coupling of additionalnucleotides to the siNA sequence. This approach allows the conjugatemoiety to be positioned anywhere within the siNA molecule.

Conjugate derivatives can also be introduced to a siNA molecule postsynthetically. Post synthetic conjugation allows a conjugate moiety tobe introduced at any position within the siNA molecule where anappropriate functional group is present (e.g., a C5 alkylamine linkerpresent on a nucleotide base or a 2′-alkylamine linker present on anucleotide sugar can provide a point of attachment for an NHS-conjugatemoiety). Generally, a reactive chemical group present in the siNAmolecule is unmasked following synthesis, thus allowing post-syntheticcoupling of the conjugate to occur. In a non-limiting example, anprotected amino linker containing nucleotide (e.g., TFA protected C5propylamino thymidine) is introduced at a desired position of the siNAduring solid phase synthesis. Following cleavage and deprotection of thesiNA, the free amine is made available for NHS ester coupling of theconjugate at the desired position within the siNA sequence, such as atthe 3′-end of the sense and/or antisense strands, the 3′ and/or 5′-endof the sense strand, or within the siNA sequence, such as in the loopportion of a single stranded hairpin siNA sequence.

A conjugate moiety can be introduced at different locations within asiNA molecule using both solid phase synthesis and post-syntheticcoupling approaches. For example, solid phase synthesis can be used tointroduce a conjugate moiety at the 5′-end of the siNA (e.g. sensestrand) and post-synthetic coupling can be used to introduce a conjugatemoiety at the 3′-end of the siNA (e.g. sense strand and/or antisensestrand). As such, a siNA sense strand having 3′ and 5′ end conjugatescan be synthesized (see for example FIG. 65). Conjugate moieties canalso be introduced in other combinations, such as at the 5′-end, 3′-endand/or loop portions of a siNA molecule (see for example FIG. 66).

Example 20: Pharmacokinetics of siNA Conjugates (FIG. 67)

Three nuclease resistant siNA molecule targeting site 1580 of hepatitisB virus (HBV) RNA were designed using Stab 7/8 chemistry (see Table IV)and a 5′-terminal conjugate moiety.

One siNA conjugate comprises a branched cholesterol conjugate linked tothe sense strand of the siNA. The “cholesterol” siNA conjugate moleculehas the structure shown below:

where T stands for thymidine, B stands for inverted deoxyabasic, Gstands for 2′-deoxy guanosine, A stands for 2′-deoxy adenosine, G standsfor 2′-O-methyl guanosine, A stands for 2′-O-methyl adenosine, u standsfor 2′-fluoro uridine, c stands for 2′-fluoro cytidine, a stands foradenosine, and s stands for phosphorothioate linkage.

Another siNA conjugate comprises a branched phospholipid conjugatelinked to the sense strand of the siNA. The “phospholipid” siNAconjugate molecule has the structure shown below:

where T stands for thymidine, B stands for inverted deoxyabasic, Gstands for 2′-deoxy guanosine, A stands for 2′-deoxy adenosine, G standsfor 2′-O-methyl guanosine, A stands for 2′-O-methyl adenosine, u standsfor 2′-fluoro uridine, c stands for 2′-fluoro cytidine, a stands foradenosine, and s stands for phosphorothioate linkage.

Another siNA conjugate comprises a polyethylene glycol (PEG) conjugatelinked to the sense strand of the siNA. The “PEG” siNA conjugatemolecule has the structure shown below:

where T stands for thymidine, B stands for inverted deoxyabasic, Gstands for 2′-deoxy guanosine, A stands for 2′-deoxy adenosine, G standsfor 2′-O-methyl guanosine, A stands for 2′-O-methyl adenosine, u standsfor 2′-fluoro uridine, c stands for 2′-fluoro cytidine, a stands foradenosine, and s stands for phosphorothioate linkage.

The Cholesterol, Phospholipid, and PEG conjugates were evaluated forpharmakokinetic properties in mice compared to a non-conjugated siNAconstruct having matched chemistry and sequence. This study wasconducted in female CD-1 mice approximately 26 g (6-7 weeks of age).Animals were housed in groups of 3. Food and water were provided adlibitum. Temperature and humidity were according to Pharmacology TestingFacility performance standards (SOP's) which are in accordance with the1996 Guide for the Care and Use of Laboratory Animals (NRC). Animalswere acclimated to the facility for at least 3 days prior toexperimentation.

Absorbance at 260 nm was used to determine the actual concentration ofthe stock solution of pre-annealed HBV siNA. An appropriate amount ofHBV siNA was diluted in sterile veterinary grade normal saline (0.9%)based on the average body weight of the mice. A small amount of theantisense (Stab 7) strand was internally labeled with gamma 32P-ATP. The32P-labeled stock was combined with excess sense strand (Stab 8) andannealed. Annealing was confirmed prior to combination with unlableddrug. Each mouse received a subcutaneous bolus of 30 mg/kg (based onduplex) and approximately 10 million cpm (specific activity ofapproximately 15 cpm/ng).

Three animals per timepoint (1, 4, 8, 24, 72, 96 h) were euthanized byCO2 inhalation followed immediately by exsanguination. Blood was sampledfrom the heart and collected in heparinized tubes. After exsanguination,animals were perfused with 10-15 mL of sterile veterinary grade salinevia the heart. Samples of liver were then collected and frozen.

Tissue samples were homogenized in a digestion buffer prior to compoundquantitation. Quantitation of intact compound was determined byscintillation counting followed by PAGE and phosphorimage analysis.Results are shown in FIG. 43. As shown in the figure, the conjugatedsiNA constructs shown vastly improved liver PK compared to theunconjugated siNA construct.

Example 21: Cell Culture of siNA Conjugates (FIG. 68)

The Cholesterol conjugates and Phospholipid conjugated siNA constructsdescribed in Example 20 above were evaluated for cell culture efficacyin a HBV cell culture system.

Transfection of HepG2 Cells with psHBV-1 and siNA

The human hepatocellular carcinoma cell line Hep G2 was grown inDulbecco's modified Eagle media supplemented with 10% fetal calf serum,2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate,25 mM Hepes, 100 units penicillin, and 100 μg/ml streptomycin. Togenerate a replication competent cDNA, prior to transfection the HBVgenomic sequences are excised from the bacterial plasmid sequencecontained in the psHBV-1 vector. Other methods known in the art can beused to generate a replication competent cDNA. This was done with anEcoRI and Hind III restriction digest. Following completion of thedigest, a ligation was performed under dilute conditions (20 gg/ml) tofavor intermolecular ligation. The total ligation mixture was thenconcentrated using Qiagen spin columns.

siNA Activity Screen and Dose Response Assay

Transfection of the human hepatocellular carcinoma cell line, Hep G2,with replication-competent HBV DNA results in the expression of HBVproteins and the production of virions. To test the efficacy of siNAconjugates targeted against HBV RNA, the Cholesterol siNA conjugate andPhospholipid siNA conjugate described in Example 12 were compared to anon-conjugated control siNA (see FIG. 68). An inverted sequence duplexwas used as a negative control for the unconjugated siNA. Dose responsestudies were performed in which HBV genomic DNA was transfected with HBVgenomic DNA with lipid at 12.5 ug/ml into Hep G2 cells. 24 hours aftertransfection with HBV DNA, cell culture media was removed and siNAduplexes were added to cells without lipid at 10 uM, 5, uM, 2.5 uM, 1uM, and 100 nm and the subsequent levels of secreted HBV surface antigen(HBsAg) were analyzed by ELISA 72 hours post treatment (see FIG. 44). Todetermine siNA activity, HbsAg levels were measured followingtransfection with siNA. Immulon 4 (Dynax) microtiter wells were coatedovernight at 4° C. with anti-HBsAg Mab (Biostride B88-95-31ad,ay) at 1gg/ml in Carbonate Buffer (Na2CO3 15 mM, NaHCO₃35 mM, pH 9.5). The wellswere then washed 4× with PBST (PBS, 0.05% Tween® 20) and blocked for 1hr at 37° C. with PBST, 1% BSA. Following washing as above, the wellswere dried at 37° C. for 30 min. Biotinylated goat ant-HBsAg (AccurateYVS1807) was diluted 1:1000 in PBST and incubated in the wells for 1 hr.at 37° C. The wells were washed 4× with PBST. Streptavidin/AlkalinePhosphatase Conjugate (Pierce 21324) was diluted to 250 ng/ml in PBST,and incubated in the wells for 1 hr. at 37° C. After washing as above,p-nitrophenyl phosphate substrate (Pierce 37620) was added to the wells,which were then incubated for 1 hour at 37° C. The optical density at405 nm was then determined. As shown in FIG. 68, the phospholipid andcholesterol conjugates demonstrate marked dose dependent inhibition ofHBsAg expression compared to the unconjugated siNA construct whendelivered to cells without any transfection agent (lipid).

Example 22: Ex Vivo Stability of siNA Constructs

Chemically modified siNA constructs were designed and synthesized inorder to improve resistance to nucleases while maintaining silencing incell culture systems. Modified strands, designated Stab 4, Stab 5, Stab7, Stab 8, and Stab 11 (Table IV), were tested in three sets of duplexesthat demonstrated a range of stability and activity. These duplexescontained differentially modified sense and antisense strands. Allmodified sense strands contain terminal 5′ and 3′ inverted abasic caps,while antisense strands possess a 3′ terminal phosphorothioate linkage.The results characterize the impact of chemical modifications onnuclease resistance in ex vivo models of the environments sampled bydrugs.

Active siNAs were assessed for their resistance to degradation in serumand liver extracts. Stability in blood will be a requirement for asystemically administered siNA, and an anti-HBV or anti-HCV siNA wouldrequire stability and activity in the hepatic intracellular environment.Liver extracts potentially provide an extreme nuclease model where manycatabolic enzymes are present. Both mouse and human systems wereassessed.

Individual strands of siNA duplexes were internally labeled with 32P andincubated as single strands or as duplex siRNAs in human or mouse serumand liver extracts. Representative data is shown in Table VI. Throughoutthe course of the experiments, constant levels of ribonuclease activitywere verified. The extent and pattern of all-RNA siNA degradation (3minute time point) did not change following preincubation of serum orliver extract at 37° C. for up to 24 hours.

The biological activity of siRNAs containing all-ribose residues hasbeen well established. The extreme instability (t1/2=0.017 hours) ofthese compounds in serum underscores the need for chemical modificationfor use in systemic therapeutic applications. The Stab 4/5 duplexmodifications provide significant stability in human and mouse serum(t1/2's=10-408 hours) and human liver extract (t1/2's=28-43 hours).

In human serum the Stab 4 strand chemistry in the context of the Stab4/5 duplex, possesses greater stability than the Stab 5 strand chemistry(t1/2=408 vs. 39 hours). This result highlights the impact terminalmodifications have on stability. A fully-modified Stab 7/11 construct(no ribonucleotides present) was generated from the Stab 4/5 constructsby substituting the ribonucleotides in all purine positions withdeoxyribonucleotides. Another fully modified construct, Stab 7/8, wasgenerated by replacing all purine positions in the antisense strand with2′-O-methyl nucleotides. This proved to be the most stable antisensestrand chemistry observed, with t1/2=816 hours in human liver extract.

The dramatic stability of Stab 8 modifications was also observed whennon-duplexed single strands were incubated in human serum and liverextract, as shown in Table VII. An approximate five-fold increase inserum stability is seen for the double stranded constructs, compared tothat observed for the individual strands. In liver extract, the siNAduplex provides even greater stability compared to the single strands.For example, the Stab 5 chemistry is greater than 100-fold more stablein the Stab 4/5 duplex relative to its stability alone.

Terminal modifications have a large impact on stability in human serum,as can be seen from a comparison of sense verses antisense stabilitiesin duplex form, and the Stab 4 and Stab 5 single-strand stabilities.Therefore, a number of 3′ antisense capping moieties on Stab 4/5chemistry duplexes were assessed for their contribution to stability inhuman serum. The structures of these modifications are shown in FIG. 22,and resultant half-lives are shown in Table VIII. A wide range ofdifferent stabilities were observed, from half-lives as short as onehour to greater than 770 hours. Thus, in the context of 2′-fluoromodified pyrimidines, 3′-exonuclease becomes the primary mode of attackon duplexes in human serum; a number of chemistries minimize this siteof attack.

These results suggest that susceptibility to 3′ exonucleases is a majorpath to degradation in the serum.

Example 23: Activity of siNA Molecules Delivered Via HydrodynamicInjection

An in vivo mouse model that utilizes hydrodynamic tail vein injection ofa replication competent HBV vector has been used to assess the activityof chemically stabilized siRNA targeted to HBV RNA. The hydrodynamicdelivery of nucleic acids in the mouse has been described by Liu et al.,1999, Gene Therapy, 6, 1258-1266, who showed that the vast majority ofthe nucleic acid is delivered to the liver by this technique. The use ofthe hydrodynamic technology to develop an HBV mouse model has beendescribed by Yang et al., 2002, PNAS, 99, 13825-13830. In thevector-based model, HBV replicates in the liver for approximately 10days, resulting in detectable levels of HBV RNA and antigens in theliver and HBV DNA and antigens in the serum.

To assess the activity of chemically stabilized siNAs against HBV,co-injection of the siNAs along with the HBV vector was done in mousestrain C57BL/J6. The HBV vector used, pWTD, is a head-to-tail dimer ofthe complete HBV genome (see for example Buckwold et al., 1996, J.Virology, 70, 5845-5851). For a 20 gram mouse, a total injection of 1.6ml containing 10 μg or 1 μg of pWTD and 100 μg of siNA duplex in saline,was injected into the tail vein within 5 seconds. For a larger mouse,the volume is scaled to maintain a level of 140% of the blood volume ofthe mouse. The injection is done using a 3 cc syringe and 27 g ½ needle.The animals were sacrificed at 72 hrs post-injection. Animals weretreated with siNA constructs and matched chemistry inverted controls.Analysis of the HBV DNA (FIG. 80) and HBsAg (FIG. 81) levels in serumwas conducted by real-time PCR and ELISA respectively. The levels of HBVRNA in the liver (FIG. 82) were analyzed by real-time RT-PCR. In aseparate experiment, analysis of HBV DNA levels in serum was carried outat 5 days and 7 days (FIG. 83) after co-injection of siNA and the HBVvector.

Example 24: Activity Screens Using Chemically Modified siNA

Two formats can be used to identify active chemically modified siNAmolecules against target nucleic acid molecules (e.g., RNA). One formatinvolves screening unmodified siNA constructs in an appropriate system(e.g., cell culture or animal models) then applying chemicalmodifications to the sequence of identified leads and rescreening themodified constructs. Another format involves direct screening ofchemically modified constructs to identify chemically modified leads(see for example the Stab 7/8 HCV screen shown in FIG. 86 and the Stab7/8 HBV screen shown FIG. 87, as described above). The latter approachcan be useful in identifying active constructs that are specific tovarious combinations of chemical modifications (e.g., Stab 1-18chemistries shown in Table V herein). Additionally, different iterationsof such chemical modifications can be assessed using active chemicallymodified leads and appropriate rules for selective active constructsgiven a particular chemistry can be established using this approach.Non-limiting examples of such activity screen are described below.

Activity Screen of Stab 7/8 Constructs Targeting Luciferase RNA

HeLa cells were co-transfected with pGF3 vector (250 ng/well), renillaluciferase vector (10 ng/well) and siNA (0.5-25 nM) using 0.5 ullipofectamine2000 per well. Twenty-four hours post-transfection, thecells were assayed for luciferase activity using the Promega DualLuciferase Assay Kit per the manufacturer's instruction. siNA constructshaving high levels of activity were identified and tested in a doseresponse assay with concentrations ranging from 0.5 to 25 nM. Resultsfor siNA constructs targeting sites 80, 237, and 1478 are shown in FIG.84 and sites 1544 and 1607 are shown in FIG. 85. As shown in theFigures, several active Stab 7/8 constructs were identified thatdemonstrate potent dose related inhibition of luciferase expression.

Activity Screen of Combination siNA Constructs Targeting HBV RNA inHepG2 Cells

The HBV HepG2 cell culture system described in Example 13 above wasutilized to evaluate the efficacy of various combinations of chemicalmodifications (Table V) in the sense strand and antisense strand of siNAmolecules as compared to matched chemistry inverted controls. Todetermine siNA activity, HbsAg levels were measured followingtransfection with siNA. Immulon 4 (Dynax) microtiter wells were coatedovernight at 4° C. with anti-HBsAg Mab (Biostride B88-95-31ad,ay) at 1gg/ml in Carbonate Buffer (Na2CO3 15 mM, NaHCO₃35 mM, pH 9.5). The wellswere then washed 4× with PBST (PBS, 0.05% Tween® 20) and blocked for 1hr at 37° C. with PBST, 1% BSA. Following washing as above, the wellswere dried at 37° C. for 30 min. Biotinylated goat ant-HBsAg (AccurateYVS1807) was diluted 1:1000 in PBST and incubated in the wells for 1 hr.at 37° C. The wells were washed 4× with PBST. Streptavidin/AlkalinePhosphatase Conjugate (Pierce 21324) was diluted to 250 ng/ml in PBST,and incubated in the wells for 1 hr. at 37° C. After washing as above,p-nitrophenyl phosphate substrate (Pierce 37620) was added to the wells,which were then incubated for 1 hour at 37° C. The optical density at450 nm was then determined. Results of the combination HBV siNA screenare shown in FIGS. 88-90. As shown in the Figures, the variouscombinations of differing sense and antisense chemistries (e.g.,sense/antisense constructs having Stab 7/8, 7/10, 7/11, 9/8, 9/10, 6/10,16/8, 16/10, 18/8, 18/10, 4/8, 4/10, 7/5, 9/5, and 9/11 chemistry)result in active siNA constructs.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I Sirna/ SEQ RPI# Aliases Sequence ID# 28443Sirna/RPI GL2 Str1 (sense)  cGuAcGcGGAAuAcuucGATT 349 2′-amino U C 28444Sirna/RPI GL2 Str2 (antisense)  ucGAAGuAuuccGcGuAcGTT 350 2′-amino U C28445 Sirna/RPI GL2 Str1 (sense)  cGuAcGcGGAAuAcuucGAuT 3512′-amino U C uT 3′end 28446 Sirna/RPI GL2 Str2 (antisense) ucGAAGuAuuccGcGuAcGuT 352 2′-amino U C uT 3′end 30051HCV-Luc:325U21 siNA 5 5′ P =  BC_(s)C_(s)C_(s)C_(s)G_(s)GGAGGUCUCGUAGAXXB 353 S + 3′ univ. base 2 +5′/3′ invAba (antisense) 30052 HCV-Luc:325U21 siNA rev 5 5′BA_(s)G_(s)A_(s)U_(s)G_(s)CUCUGGAGGGCCCCXXB 354 P = S + 3′univ. base 2 + 5′/3′ invAba (antisense) 30053HCV-Luc:345L21 siNA (325C)  U_(s)C_(s)U_(s)A_(s)C_(s)GAGACCUCCCGGGGXXB355 (antisense) 5 5′ P = S + 3′ univ.   base 2 + 3′ invAba (sense) 30054HCV-Luc:345L21 siNA (325C)  G_(s)G_(s)G_(s)G_(s)C_(s)CCUCCAGAGCAUCUXXB356 (antisense) rev 5 5′ P = S + 3′    univ. base 2 + 3′ invAba (sense)30055 HCV-Luc:325U21 siNA all Y P =  BC_(s)C_(s)C_(s)C_(s)GGGAGGU_(s)C_(s)U_(s)C_(s)GU_(s)AGAXXB 357 S + 3′univ. base 2 + 5′/3′ invAba (antisense) 30056HCV-Luc:325U21 siNA rev all BAGAU_(s)GC_(s)U_(s)C_(s)U_(s)GGAGGGC_(s)C_(s)C_(s)C_(s)XXB 358 Y P =S + 3′ univ. base 2 + 5′/3′ invAba (antisense) 30057HCV-Luc:345L21 siNA (325C) U_(s)C_(s)U_(s)AC_(s)GAGAC_(s)C_(s)U_(s)C_(s)C_(s)C_(s)GGGGXXB 359(antisense) all Y P = S + 3′     univ. base 2 + 3′ invAba  (sense) 30058HCV-Luc:345L21 siNA (325C) GGGGC_(s)C_(s)C_(s)U_(s)C_(s)C_(s)AGAGC_(s)AU_(s)C_(s)U_(s)XXB 360(antisense) rev all Y P = S +     3′ univ. base 2 + 3′ invAba  (sense)30059 HCV-Luc:325U21 siNA 4/3 P = S Bc_(s)c_(s)c_(s)c_(s)GGGAGGucucGuA_(s)G_(s)A_(s)XXB 361 ends +all Y-2′F + 3′ univ. base   2 + 5′/3′ invAba (antisense) 30060HCV-Luc:325U21 siNA rev 4/3 P = S BA_(s)G_(s)A_(s)u_(s)GcucuGGAGGGcc_(s)c_(s)c_(s)XXB 362 ends +all Y-2′F + 3′ univ. base    2 + 5′/3′ invAba (antisense) 30170HCV-Luc:325U21 siNA all Y-2′F + B ccccGGGAGGucucGuAGAXX B 363 3′univ. base 2 + 5′/3′ invAba  (antisense) 30171HCV-Luc:325U21 siNA rev all  B AGAuGcucuGGAGGGccccXX B 364 Y-2′F + 3′univ. base 2 +   5′/3′ invAba (antisense) 30172HCV-Luc:345L21 siNA (325C) B U_(s)C_(s)U_(s)AC_(s)GAGAC_(s)C_(s)U_(s)C_(s)C_(s)C_(s)GGGGXX B 365(antisense) all Y P = S + 3′ univ. base 2 + 5′/3′ invAba (antisense)30173 HCV-Luc:345L21 siNA (325C)  ucuAcGAGAccucccGGGG 366(antisense) all Y-2′F 30174 HCV-Luc:345L21 siNA (325C) GGGGcccuccAGAGcAucu 367 (antisense) rev all Y-2′F 30175HCV-Luc:345L21 siNA (325C)  ucuAcGAGAccucccGGGGXX 368(antisense) all Y-2′F + 3′ univ. base 2 30176HCV-Luc:345L21 siNA (325C)  GGGGcccuccAGAGcAucuXX 369(antisense) rev all Y-2′F + 3′ univ. base 2 30177HCV-Luc:345L21 siNA (325C)  B ucuAcGAGAccucccGGGGXX B 370(antisense) all Y-2′F + 3′ univ.   base 2 + 5′/3′ iB 30178HCV-Luc:325U21 siNA all Y P =  C_(s)C_(s)C_(s)C_(s)GGGAGGU_(s)C_(s)U_(s)C_(s)GU_(s)AGAXX B 371 S + 3′univ. base 2 + 3′ invAba  (sense) 30063 Sirna/RPI GL2 Str1 (sense)   BcGuAcGcGGAAuAcuucGATTB 372 2′-F U, C + 3′, 5′ abasic 30222Sirna/RPI GL2 Str1 (sense) Y  B cGuAcGcGGAAuAcuucGATT B 3732′-O-Me with 3′-TT & 5′/3′ iB 30224 Sirna/RPI GL2 Str2 (antisense) Y  ucGAAGuAuuccGcGuAcGT_(s)T 374 2′-F & 3′ TsT 30430Sirna/RPI GL2 Str2 (antisense)    ucgaaguauuccgcguacgT_(s)T 3752′-F U, C + 5′, 3′ abasic, A,  G = 2′-O-Me 30431Sirna/RPI GL2 Str1 (sense)    BcguacgcggaauacuucgaTTB 376 2′-F U, C +3′, 5′ abasic, TT;  2′-O-Me-A, G 30433 Sirna/RPI GL2 Str1 (sense)   BcGuAcGcGGAAuAcuucGATTB 377 2′-F U, C + 3′, 5′ abasic, TT; 2′-deoxy-A, G 30550 Sirna/RPI GL2 Str2 (antisense) ucGAAGuAuuccGcGuAcGT_(s)t 378 2′-F U, C 3′-dTsT 30555Sirna/RPI GL2 Str2 (antisense)  ucGAAGuAuuccGcGuAcGTL 3792′-F U, C 3′-glycerol.T 30556 Sirna/RPI GL2 Str2 (antisense) ucGAAGuAuuccGcGuAcGTTL 380 2′-F U, C 3′-glycerol, 2T 30226rev Sirna/RPI GL2 Str1 (sense)  B AGcuucAuAAGGcGcAuGcTT B 381Y 2′-O-Me with 3′-TT & 5′/3′ iB 30227 rev Sirna/RPI GL2 Str1 (sense) B AGcuucAuAAGGcGcAuGcTT B 382 Y 2′-F with 3′-TT & 5′/3′ iB 30229rev Sirna/RPI GL2 Str2   GcAuGcGccuuAuGAAGcuT_(s)T 383(antisense) Y 2′-F & 3′ TsT 30434 Sirna/RPI GL2 Str1 (sense) BcguacgcGGAAuAcuucgaTTB 384 2′-F U, C + 3′, 5′ Abasic,TT; 2′-O-Me-A, G; ribo core 30435 Sirna/RPI GL2 Str1 (sense) BcGuAcGcGGAAuAcuucGATTB 385 2′-F U, C + 3′, 5′ Abasic,TT; 2′-deoxyA, G; ribo core 30546 Sirna/RPI GL2 Str2 (antisense) ucGAAGuAuuccGcGuAcG3T 386 2′-F U, C 3′-dTT 30551Sirna/RPI GL2 Str2 (antisense)  ucGAAGuAuuccGcGuAcGTddC 3872′-F U, C dTddC 30557 Sirna/RPI GL2 Str2 (antisense) ucGAAGuAuuccGcGuAcGT 388 2′-F U, C 3′-invertedT, T 30558Sirna/RPI GL2 Str2 (antisense)  ucGAAGuAuuccGcGuAcGTT 3892′-F U, C 3′-invertedT, TT 30196 FLT1:2340U21 siRNA sense iB capsB cAAccAcAAAAuAcAAcAATT B 419 w/2′FY's 30416 FLT1:2358L21 siRNA (2340C) uuGuuGuAuuuuGuGGuuGT_(s)T 420 (antisense) TsT 29548HBV:394L21 siRNA (414C)  GAUGAGGCAUAGCAGCAGGTT 421 (antisense) 29544HBV:414U21 siRNA pos (sense) CCUGCUGCUAUGCCUCAUCTT 422 29556HBV:394L21 siRNA neg (414C)  GGACGACGAUACGGAGUAGTT 423 (antisense) inv29552 HBV:414U21 siRNA pos (sense) inv CUACUCCGUAUCGUCGUCCTT 424 30350HBV:262U21 siRNA stab04 (sense) B uGGAcuucucucAAuuuucuA B 425 30361HBV:280L21 siRNA (262C)  GAAAAuuGAGAGAAGuccAT_(s)T 426(antisense) stab05 30372 HBV:262U21 siRNA inv stab04 B AucuuuuAAcucucuucAGGu B 427 (sense) 30383 HBV:280L21 siRNA (262C)  AccuGAAGAGAGuuAAAAGT_(s)T 428 (antisense) inv stab05 30352HBV:380U21 siRNA stab04 (sense) B uGuGucuGcGGcGuuuuAucA B 429 30363HBV:398L21 siRNA (380C)  AuAAAAcGccGcAGAcAcAT_(s)T 430(antisense) stab05 30374 HBV:380U21 siRNA inv stab04 B AcuAuuuuGcGGcGucuGuGu B 431 (sense) 30385 HBV:398L21 siRNA (380C)  AcAcAGAcGccGcAAAAuAT_(s)T 432 (antisense) inv stab05 30353HBV:413U21 siRNA stab04 (sense) B uccuGcuGcuAuGccucAucu B 433 30364HBV:431L21 siRNA (413C)  AuGAGGcAuAGcAGcAGGAT_(s)T 434(antisense) stab05 30375 HBV:413U21 siRNA inv stab04 B ucuAcuccGuAucGucGuccu B 435 (sense) 30386 HBV:431L21 siRNA (413C)  AGGAcGAcGAuAcGGAGuAT_(s)T 436 (antisense) inv stab05 30354HBV:462U21 siRNA stab04 (sense) B uAuGuuGcccGuuuGuccucu B 437 30365HBV:480L21 siRNA (462C)  AGGAcAAAcGGGcAAcAuAT_(s)T 438(antisense) stab05 30376 HBV:462U21 siRNA inv stab04 B ucuccuGuuuGcccGuuGuAu B 439 (sense) 30387 HBV:480L21 siRNA (462C)  AuAcAAcGGGcAAAcAGGAT_(s)T 440 (antisense) inv 30355HBV:1580U21 siRNA stab04 (sense) B uGuGcAcuucGcuucAccucu B 441 30366HBV:1598L21 siRNA (1580C)  AGGuGAAGcGAAGuGcAcAT_(s)T 442(antisense) stab05 30377 HBV:1580U21 siRNA inv stab04 B ucuccAcuucGcuucAcGuGu B 443 (sense) 30388 HBV:1598L21 siRNA (1580C) AcAcGuGAAGcGAAGuGGAT_(s)T 444 (antisense) inv stab05 30356HBV:1586U21 siRNA stab04 (sense) B cuucGcuucAccucuGcAcGu B 445 30367HBV:1604L21 siRNA (1586C)  GuGcAGAGGuGAAGcGAAGT_(s)T 446(antisense) stab05 30378 HBV:1586U21 siRNA inv stab04 B uGcAcGucuccAcuucGcuuc B 447 (sense) 30389 HBV:1604L21 siRNA (1586C) GAAGcGAAGuGGAGAcGuGT_(s)T 448 (antisense) inv stab05 30357HBV:1780U21 siRNA stab04 (sense) B AGGcuGuAGGcAuAAAuuGGu B 449 30368HBV:1798L21 siRNA (1780C)  cAAuuuAuGccuAcAGccuT_(s)T 450(antisense) stab05 30379 HBV:1780U21 siRNA inv stab04 B uGGuuAAAuAcGGAuGucGGA B 451 (sense) 30390 HBV:1798L21 siRNA (1780C) uccGAcAuccGuAuuuAAcT_(s)T 452 (antisense) inv stab05 30612HBV:1580U21 siRNA stab07 (sense) B uGuGcAcuucGcuucAccuTT B 453 30620HBV:1598L21 siRNA (1580C)  aggugaagcgaagugcacaT_(s)T 454(antisense) stab08 30628 HBV:1582U21 siRNA inv stab07 B ucuccAcuucGcuucAcGuTT B 455 (sense) 30636 HBV:1596L21 siRNA (1578C) gcacacgugaagcgaagugT_(s)T 456 (antisense) inv stab08 30612HBV:1580U21 siRNA stab07 (sense) B uGuGcAcuucGcuucAccuTT B 457 31175HBV:1598L21 siRNA (1580C) stab11 AGGuGAAGcGAAGuGcAcAT_(s)T 458(antisense) 30612 HBV:1580U21 siRNA stab07 (sense)B uGuGcAcuucGcuucAccuTT B 459 31176 HBV:1596L21 siRNA (1578C) GcAcAcGuGAAGcGAAGuGT_(s)T 460 (antisense) inv stab11  (antisense) 30287HBV:1580U21 siRNA (sense) UGUGCACUUCGCUUCACCUCU 461 30298HBV:1598L21 siRNA (1580C)  AGGUGAAGCGAAGUGCACACG 462 (antisense) 30355HBV:1580U21 siRNA stab04 (sense) B uGuGcAcuucGcuucAccucu B 463 30366HBV:1598L21 siRNA (1580C)  AGGuGAAGcGAAGuGcAcATsT 464 (antisense) stab0530612 HBV:1580U21 siRNA stab07 (sense) B uGuGcAcuucGcuucAccuTT B 46531175 HBV:1598L21 siRNA (1580C) stab11 AGGuGAAGcGAAGuGcAcATsT 466(antisense) 30612 HBV:1580U21 siRNA stab07 (sense)B uGuGcAcuucGcuucAccuTT B 467 30620 HBV:1598L21 siRNA (1580C) AGGuGAAGcGAAGuGcAcATsT 468 (antisense) stab08 31335HBV:1580U21 siRNA stab09 (sense) B UGUGCACUUCGCUUCACCUTT B 469 31337HBV:1598L21 siRNA (1580C) stab10 AGGUGAAGCGAAGUGCACATsT 470 (antisense)31456 HCVa:291U21 siRNA stab04 B cuuGuGGuAcuGccuGAuATT B 471 31468HCVa:309L21 siRNA (291C) stab05 uAucAGGcAGuAccAcAAGTsT 472 31480HCVa:291U21 siRNA inv stab04 B AuAGuccGucAuGGuGuucTT B 473 31492HCVa:309L21 siRNA (291C) inv  GAAcAccAuGAcGGAcuAuTsT 474 stab05 31461HCVa:300U21 siRNA stab04 B cuGccuGAuAGGGuGcuuGTT B 475 31473HCVa:318L21 siRNA (300C) stab05 cAAGcAcccuAucAGGcAGTsT 476 31485HCVa:300U21 siRNA inv stab04 B GuucGuGGGAuAGuccGucTT B 477 31497HCVa:318L21 siRNA (300C) inv  GAcGGAcuAucccAcGAAcTsT 478 stab05 31463HCVa:303U21 siRNA stab04 B ccuGAuAGGGuGcuuGcGATT B 479 31475HCVa:321L21 siRNA (303C) stab05 ucGcAAGcAcccuAucAGGTsT 480 31487HCVa:303U21 siRNA inv stab04 B AGcGuucGuGGGAuAGuccTT B 481 31499HCVa:321L21 siRNA (303C) inv  GGAcuAucccAcGAAcGcuTsT 482 stab05 31344HCVa:325U21 siRNA stab07 B ccccGGGAGGucucGuAGATT B 483 30562HCVa:345L21 siRNA (325C) Y-2′F, ucuAcGAGAccucccGGGGTsT 484  R-2′OMe +TsT 31345 HCVa:325U21 siRNA inv stab07 B AGAuGcucuGGAGGGccccTT B 48531346 HCVa:343L21 siRNA (325C) inv  GGGGcccuccAGAGcAucuTsT 486 stab0831702 HCVa:326U21 siRNA stab07 B cccGGGAGGucucGuAGAcTT B 487 31706HCVa:344L21 siRNA (326C) stab08 GucuAcGAGAccucccGGGTsT 488 31710HCVa:326U21 siRNA inv stab07 B cAGAuGcucuGGAGGGcccTT B 489 31714HCVa:344L21 siRNA (326C) inv  GGGcccuccAGAGcAucuGTsT 490 stab08 31703HCVa:327U21 siRNA stab07 B ccGGGAGGucucGuAGAccTT B 491 31707HCVa:345L21 siRNA (327C) stab08 GGucuAcGAGAccucccGGTsT 492 31711HCVa:327U21 siRNA inv stab07 B ccAGAuGcucuGGAGGGccTT B 493 31715HCVa:345L21 siRNA (327C) inv  GGcccuccAGAGcAucuGGTsT 494 stab08 31704HCVa:328U21 siRNA stab07 B cGGGAGGucucGuAGAccGTT B 495 31708HCVa:346L21 siRNA (328C) stab08 cGGucuAcGAGAccucccGTsT 496 31712HCVa:328U21 siRNA inv stab07 B GccAGAuGcucuGGAGGGcTT B 497 31716HCVa:346L21 siRNA (328C) inv  GcccuccAGAGcAucuGGcTsT 498 stab08 31705HCVa:329U21 siRNA stab07 B GGGAGGucucGuAGAccGuTT B 499 31709HCVa:347L21 siRNA (329C) stab08 AcGGucuAcGAGAccucccTsT 500 31713HCVa:329U21 siRNA inv stab07 B uGccAGAuGcucuGGAGGGTT B 501 31717HCVa:347L21 siRNA (329C) inv  cccuccAGAGcAucuGGcATsT 502 stab08 31703HCVa:327U21 siRNA stab07 B ccGGGAGGucucGuAGAccTT B 503 31707HCVa:345L21 siRNA (327C) stab08 GGucuAcGAGAccucccGGTsT 504 31711HCVa:327U21 siRNA inv stab07 B ccAGAuGcucuGGAGGGccTT B 505 31715HCVa:345L21 siRNA (327C) inv  GGcccuccAGAGcAucuGGTsT 506 stab08 29579HCVa:325U21 siRNA CCCCGGGAGGUCUCGUAGACCGU 543HCVa:327 siRNA 3′-classI 10 bp UCUCGUAGACCUUGGUCUACGAGACCUCCCGGTT 544HCVa:327 siRNA 3′-classI 8 bp UCGUAGACCUUGGUCUACGAGACCUCCCGGTT 545HCVa:327 siRNA 3′-classI 6 bp GUAGACCUUGGUCUACGAGACCUCCCGGTT 546HCVa:327 siRNA 3′-classI 4 bp AGACCUUGGUCUACGAGACCUCCCGGTT 547HCVa:327 siRNA 5′-classI 10 bp GGUCUACGAGACCUCCCGGUUCCGGGAGGUCU 548HCVa:327 siRNA 5′-classI 8 bp GGUCUACGAGACCUCCCGGUUCCGGGAGGU 549HCVa:327 siRNA 5′-classI 6 bp GGUCUACGAGACCUCCCGGUUCCGGGAG 550HCVa:327 siRNA 5′-classI 4 bp GGUCUACGAGACCUCCCGGUUCCGGG 551HCVa:327 siRNA 3′-gaaa 10 bp CUCGUAGACCGAAAGGUCUACGAGACCUCCCGGTT 552HCVa:327 siRNA 3′-gaaa 8 bp CGUAGACCGAAAGGUCUACGAGACCUCCCGGTT 553HCVa:327 siRNA 3′-gaaa 6 bp UAGACCGAAAGGUCUACGAGACCUCCCGGTT 554HCVa:327 siRNA 3′-gaaa 4 bp GACCGAAAGGUCUACGAGACCUCCCGGTT 555HCVa:327 siRNA 5′-gaaa 10 bp GGUCUACGAGACCUCCCGGUUGAAACCGGGAGGUC 556HCVa:327 siRNA 5′-gaaa 8 bp GGUCUACGAGACCUCCCGGUUGAAACCGGGAGG 557HCVa:327 siRNA 5′-gaaa 6 bp GGUCUACGAGACCUCCCGGUUGAAACCGGGA 558HCVa:327 siRNA 5′-gaaa 4 bp GGUCUACGAGACCUCCCGGUUGAAACCGG 559HCVa:327 siRNA 3′-uuuguguag 10 bpCGUAGACCUUUUUGUGUAGGGUCUACGAGACCUCCCGGTT 560HCVa:327 siRNA 3′-uuuguguag 8 bp UAGACCUUUUUGUGUAGGGUCUACGAGACCUCCCGGTT561 HCVa:327 siRNA 3′-uuuguguag 6 bpGACCUUUUUGUGUAGGGUCUACGAGACCUCCCGGTT 562HCVa:327 siRNA 3′-uuuguguag 4 bp CCUUUUUGUGUAGGGUCUACGAGACCUCCCGGTT 563HCVa:327 siRNA 5′-uuuguguag 10 bpGGUCUACGAGACCUCCCGGUUUUUGUGUAGCCGGGAGGUC 564HCVa:327 siRNA 5′-uuuguguag 8 bp GGUCUACGAGACCUCCCGGUUUUUGUGUAGCCGGGAGG565 HCVa:327 siRNA 5′-uuuguguag 6 bpGGUCUACGAGACCUCCCGGUUUUUGUGUAGCCGGGA 566HCVa:327 siRNA 5′-uuuguguag 4 bp GGUCUACGAGACCUCCCGGUUUUUGUGUAGCCGG 567HCVa:327 siRNA 3′-classI 10 bp  ucucGuAGAccuuGGucuAcGAGAccucccGGTsT 568stab08 HCVa:327 siRNA 3′-classI 8 bp  ucGuAGAccuuGGucuAcGAGAccucccGGTsT569 stab08 HCVa:327 siRNA 3′-classI 6 bp GuAGAccuuGGucuAcGAGAccucccGGTsT 570 stab08HCVa:327 siRNA 3′-classI 4 bp  AGAccuuGGucuAcGAGAccucccGGTsT 571 stab08HCVa:327 siRNA 5′-classI 10 bp  GGucuAcGAGAccucccGGuuccGGGAGGucu 572stab08 HCVa:327 siRNA 5′-classI 8 bp  GGucuAcGAGAccucccGGuuccGGGAGGu 573stab08 HCVa:327 siRNA 5′-classI 6 bp  GGucuAcGAGAccucccGGuuccGGGAG 574stab08 HCVa:327 siRNA 5′-classI 4 bp  GGucuAcGAGAccucccGGuuccGGG 575stab08 HCVa:327 siRNA 3′- gaaa 10 bp cucGuAGAccGAAAGGucuAcGAGAccucccGGTsT 576 stab08HCVa:327 siRNA 3′- gaaa 8 bp  cGuAGAccGAAAGGucuAcGAGAccucccGGTsT 577stab08 HCVa:327 siRNA 3′-gaaa 6 bp  uAGAccGAAAGGucuAcGAGAccucccGGTsT 578stab08 HCVa:327 siRNA 3′-gaaa 4 bp  GAccGAAAGGucuAcGAGAccucccGGTsT 579stab08 HCVa:327 siRNA 5′-gaaa 10 bp  GGucuAcGAGAccucccGGuuGAAAccGGGAGGuc580 stab08 HCVa:327 siRNA 5′-gaaa 8 bp GGucuAcGAGAccucccGGuuGAAAccGGGAGG 581 stab08HCVa:327 siRNA 5′-gaaa 6 bp  GGucuAcGAGAccucccGGuuGAAAccGGGA 582 stab08HCVa:327 siRNA 5′-gaaa 4 bp  GGucuAcGAGAccucccGGuuGAAAccGG 583 stab08HCVa:327 siRNA 3′-uuuguguag 10 bpcGuAGAccuuuuuGuGuAGGGucuAcGAGAccucccGGTsT 584 stab08HCVa:327 siRNA 3′-uuuguguag 8 bp uAGAccuuuuuGuGuAGGGucuAcGAGAccucccGGTsT585 stab08 HCVa:327 siRNA 3′-uuuguguag 6 bpGAccuuuuuGuGuAGGGucuAcGAGAccucccGGTsT 586 stab08HCVa:327 siRNA 3′-uuuguguag 4 bp ccuuuuuGuGuAGGGucuAcGAGAccucccGGTsT 587stab08 HCVa:327 siRNA 5′-uuuguguag 10 bpGGucuAcGAGAccucccGGuuuuuGuGuAGccGGGAGGuc 588 stab08HCVa:327 siRNA 5′-uuuguguag 8 bp GGucuAcGAGAccucccGGuuuuuGuGuAGccGGGAGG589 stab08 HCVa:327 siRNA 5′-uuuguguag 6 bpGGucuAcGAGAccucccGGuuuuuGuGuAGccGGGA 590 stab08HCVa:327 siRNA 5′-uuuguguag 4 bp GGucuAcGAGAccucccGGuuuuuGuGuAGccGG 591stab08 HCVa:347L23 siRNA (327C) stab08 AcGGucuAcGAGAccucccGGTsT 592HCVa:346L22 siRNA (327C) stab08 cGGucuAcGAGAccucccGGTsT 593HCVa:345L21 siRNA (327C) stab08 GGucuAcGAGAccucccGGTsT 594HCVa:344L20 siRNA (327C) stab08 GucuAcGAGAccucccGGTsT 595HCVa:343L19 siRNA (327C) stab08 ucuAcGAGAccucccGGTsT 596HCVa:342L18 siRNA (327C) stab08 cuAcGAGAccucccGGTsT 597HCVa:341L17 siRNA (327C) stab08 uAcGAGAccucccGGTsT 598HCVa:340L16 siRNA (327C) stab08 AcGAGAccucccGGTsT 599HCVa:339L15 siRNA (327C) stab08 cGAGAccucccGGTsT 600HCVa:345L21 siRNA (327C) stab08  GGucuAcGAGAccucccGGGsG 601 GGHCVa:345L20 siRNA (327C) stab08  GGucuAcGAGAccucccGGsG 602 GHCVa:345L20 siRNA (327C) stab08 GGucuAcGAGAccucccGGsT 603HCVa:345L19 siRNA (327C) stab08 GGucuAcGAGAccucccGsG 604HCVa:345L18 siRNA (327C) stab08 GGucuAcGAGAccucccsG 605HCVa:345L17 siRNA (327C) stab08 GGucuAcGAGAccuccsc 606HCVa:345L16 siRNA (327C) stab08 GGucuAcGAGAccucsc 607HCVa:345L15 siRNA (327C) stab08 GGucuAcGAGAccusc 608HCVa:327U21 siRNA stab07 B ccGGGAGGucucGuAGAccTT B 609HCVa:327U21 siRNA stab07 GT B ccGGGAGGucucGuAGAccGT B 610HCVa:327U21 siRNA stab07 B cGGGAGGucucGuAGAccTT B 611HCVa:328U20 siRNA stab07 B GGGAGGucucGuAGAccTT B 612HCVa:329U19 siRNA stab07 B GGAGGucucGuAGAccTT B 613HCVa:330U18 siRNA stab07 B GAGGucucGuAGAccTT B 614HCVa:331U17 siRNA stab07 B AGGucucGuAGAccTT B 615HCVa:332U16 siRNA stab07 B ccGGGAGGucucGuAGAccT B 616HCVa:327U21 siRNA stab07 B ccGGGAGGucucGuAGAcc B 617HCVa:327U21 siRNA stab07 B ccGGGAGGucucGuAGAc B 618HCVa:327U21 siRNA stab07 B ccGGGAGGucucGuAGA B 619HCVa:327U21 siRNA stab07 B ccGGGAGGucucGuAG B 620 31270FLT1:349U21 siRNA stab09 sense B CUGAGUUUAAAAGGCACCCTT B 621 31273FLT1:367L21 siRNA (349C) stab10 GGGUGCCUUUUAAACUCAGTsT 622 antisense31276 FLT1:349U21 siRNA stab09 inv  B CCCACGGAAAAUUUGAGUCTT B 623 sense31279 FLT1:367L21 siRNA (349C) stab10  GACUCAAAUUUUCCGUGGGTsT 624inv antisense 31679 HBV1598 all RNA sense AGGUGAAGCGAAGUGCACAUU 62530287 HBV1598 all RNA antisense UGUGCACUUCGCUUCACCUCU 626 31336HBV:1580U21 siRNA inv stab09  B UCCACUUCGCUUCACGUGUTT B 627 sense 31338HBV:1598L21 siRNA (1580C) inv  ACACGUGAAGCGAAGUGGATsT 629stab10 antisense 32636 Luc3:80U21 siRNA stab07 senseB AuAAGGcuAuGAAGAGAuATT B 630 32676 Luc3:98L21 siRNA (80C) stab08 uAucucuucAuAGccuuAuTsT 631 antisense 32640Luc3:237U21 siRNA stab07 sense B cGuAuGcAGuGAAAAcucuTT B 632 32680Luc3:255L21 siRNA (237C) stab08 AGAGuuuucAcuGcAuAcGTsT 633 antisense32662 Luc3:1478U21 siRNA stab07 sense B uGAcGGAAAAAGAGAucGuTT B 63432702 Luc3:1496L21 siRNA (1478C) stab08 AcGAucucuuuuuccGucATsT 635antisense 32666 Luc3:1544U21 siRNA stab07 senseB GAGuuGuGuuuGuGGAcGATT B 636 32706 Luc3:1562L21 siRNA (1544C) stab08ucGuccAcAAAcAcAAcucTsT 637 antisense 32672Luc3:1607U21 siRNA stab07 sense B GAGAGAuccucAuAAAGGcTT B 638 32712Luc3:1625L21 siRNA (1607C) stab08 GccuuuAuGAGGAucucucTsT 639 antisense33139 HCVa:282U21 siRNA stab07 sense B GcGAAAGGccuuGuGGuAcTT B 640 33179HCVa:300L21 siRNA (282C) stab08 GuAccAcAAGGccuuucGcTsT 641 antisense33140 HCVa:283U21 siRNA stab07 sense B cGAAAGGccuuGuGGuAcuTT B 642 33180HCVa:301L21 siRNA (283C) stab08 AGuAccAcAAGGccuuucGTsT 643 antisense33145 HCVa:289U21 siRNA stab07 sense B GccuuGuGGuAcuGccuGATT B 644 33185HCVa:307L21 siRNA (289C) stab08 ucAGGcAGuAccAcAAGGcTsT 645 antisense33149 HCVa:304U21 siRNA stab07 sense B cuGAuAGGGuGcuuGcGAGTT B 646 33183HCVa:304L21 siRNA (286C) stab08 GGcAGuAccAcAAGGccuuTsT 647 antisense33150 HCVa:305U21 siRNA stab07 sense B uGAuAGGGuGcuuGcGAGuTT B 648 33190HCVa:323L21 siRNA (305C) stab08 AcucGcAAGcAcccuAucATsT 649 antisense33151 HCVa:307U21 siRNA stab07 sense B AuAGGGuGcuuGcGAGuGcTT B 650 33191HCVa:325L21 siRNA (307C) stab08 GcAcucGcAAGcAcccuAuTsT 651 antisense33158 HCVa:317U21 siRNA stab07 sense B uGcGAGuGccccGGGAGGuTT B 652 33187HCVa:317L21 siRNA (299C) stab08 AAGcAcccuAucAGGcAGuTsT 653 antisense33210 HBV:258U21 siRNA stab07 sense B GuGGuGGAcuucucucAAuTT B 654 33250HBV:276L21 siRNA (258C) stab08 AuuGAGAGAAGuccAccAcTsT 655 antisense33212 HBV:260U21 siRNA stab07 sense B GGuGGAcuucucucAAuuuTT B 656 33252HBV:278L21 siRNA (260C) stab08 AAAuuGAGAGAAGuccAccTsT 657 antisense33214 HBV:263U21 siRNA stab07 sense B GGAcuucucucAAuuuucuTT B 658 33254HBV:281L21 siRNA (263C) stab08 AGAAAAuuGAGAGAAGuccTsT 659 antisense32429 HBV:1583U21 siRNA stab07 sense B GcAcuucGcuucAccucuGTT B 660 32438HBV:1601L21 siRNA (1583C) stab08 cAGAGGuGAAGcGAAGuGcTsT 661 antisense33226 HBV:1585U21 siRNA stab07 sense B AcuucGcuucAccucuGcATT B 662 33266HBV:1603L21 siRNA (1585C) stab08 uGcAGAGGuGAAGcGAAGuTsT 663 antisense31651 HBV:1580U21 siRNA stab06 sense B UGUGCACUUCGCUUCACCUTT B 664 31652HBV:1580U21 siRNA inv stab06  B UCCACUUCGCUUCACGUGUTT B 665 sense 31653HBV:1580U21 siRNA stab16 sense B UGUGCACUUCGCUUCACCUTT B 666 31654HBV:1580U21 siRNA inv stab16  B UCCACUUCGCUUCACGUGUTT B 667 sense 31657HBV:1580U21 siRNA stab18 sense B uGuGcAcuucGcuucAccuTT B 668 31658HBV:1580U21 siRNA inv stab18  B uccAcuucGcuucAcGuGuTT B 669 sense UPPERCASE = ribonucleotide UPPER CASE UNDERLINE = 2′-O-methyl nucleotideLowercase = 2′-deoxy-2′-fluoro nucleotide T = thymidine T = invertedthymidine t = 3′-deoxy thymidine B = inverted deoxyabasic succinatelinker B = inverted deoxyabasic X = universal base (5-nitroindole) Z =universal base (3-nitropyrrole) S = phosphorothioate internucleotidelinkage U = 5-bromodeoxyuridine A = deoxyadenosine G = deoxyguanosine L= glyceryl moiety ddC = dideoxy Cytidine p = phosphate

TABLE II Reagent Equivalents Amount Wait Time* DNA Wait Time*2′-O-methyl Wait Time*RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec  2.5 min  7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec  2.5 min  7.5 min Acetic Anhydride 100 233 μL  5 sec 5 sec  5 sec N-Methyl 186 233 μL  5 sec  5 sec  5 sec Imidazole TCA 176 2.3 mL 21 sec 21 sec 21 sec Iodine 11.2  1.7 mL 45 sec 45 sec 45 secBeaucage 12.9 645 μL 100 sec  300 sec  300 sec  Acetonitrile NA 6.67 mLNA NA NA B. 0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites15  31 μL 45 sec 233 sec  465 sec  S-Ethyl Tetrazole 38.7  31 μL 45 sec233 min 465 sec  Acetic Anhydride 655 124 μL  5 sec  5 sec  5 secN-Methyl 1245 124 μL  5 sec  5 sec  5 sec Imidazole TCA 700 732 μL 10sec 10 sec 10 sec Iodine 20.6 244 μL 15 sec 15 sec 15 sec Beaucage 7.7232 μL 100 sec  300 sec  300 sec  Acetonitrile NA 2.64 mL NA NA NA C.0.2 μmol Synthesis Cycle 96 well Instrument Equivalents: DNA/2′- Amount:DNA/2′-O- Wait Wait Time* 2′- Wait Reagent O-methyl/Ribo methyl/RiboTime* DNA O-methyl Time* Ribo Phosphoramidites 22/33/66 40/60/120 μL  60sec 180 sec  360 sec  S-Ethyl Tetrazole 70/105/210 40/60/120 μL  60 sec180 min 360 sec  Acetic Anhydride 265/265/265 50/50/50 μL 10 sec 10 sec10 sec N-Methyl 502/502/502 50/50/50 μL 10 sec 10 sec 10 sec ImidazoleTCA 238/475/475 250/500/500 μL   15 sec 15 sec 15 sec Iodine 6.8/6.8/6.880/80/80 μL 30 sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec  200 sec  Acetonitrile NA 1150/1150/1150 μL    NA NA NA • Waittime does not include contact time during delivery. • Tandem synthesisutilizes double coupling of linker molecule

TABLE III Number Solution on Stock VEGF of Injectate Conc. Group Filter(1.0 μL) concentration Animals (6.0 μL) Dose Injectate 1 Tris-Cl pH 6.9NA 5 water NA NA 2 R&D Systems 3.53 μg/μL 5 water NA NA VEGF-carrierfree 75 μM 3 R&D Systems 3.53 μg/μL 5 Site 2340 10 μg/eye   1.67 μg/μLVEGF-carrier Stab1 free siRNA 75 μM 4 R&D Systems 3.53 μg/μL 5 Site 23403 μg/eye  0.5 μg/μL VEGF-carrier Stab1 free siRNA 75 μM 5 R&D Systems3.53 μg/μL 5 Site 2340 1 μg/eye 0.167 μg/μL VEGF-carrier Stab1 freesiRNA 75 μM 6 R&D Systems 3.53 μg/μL 5 Inactive 10 μg/eye   1.67 μg/μLVEGF-carrier Site 2340 free Stab1 75 μM siRNA 7 R&D Systems 3.53 μg/μL 5Inactive 3 μg/eye  0.5 μg/μL VEGF-carrier Site 2340 free Stab1 75 μMsiRNA 8 R&D Systems 3.53 μg/μL 5 Inactive 1 μg/eye 0.167 μg/μLVEGF-carrier Site 2340 free Stab1 75 μM siRNA

TABLE IV Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 1” Ribo Ribo — 5 at 5′-end S/AS 1 at 3′-end “Stab 2” RiboRibo — All Usually AS linkages “Stab 3” 2′-fluoro Ribo — 4 at 5′-endUsually S 4 at 3′-end “Stab 4” 2′-fluoro Ribo 5′ and 3′- — Usually Sends “Stab 5” 2′-fluoro Ribo — 1 at 3′-end Usually AS “Stab 6” 2′-O-Ribo 5′ and 3′- — Usually S Methyl ends “Stab 7” 2′-fluoro 2′-deoxy 5′and 3′- — Usually S ends “Stab 8” 2′-fluoro 2′-O- — 1 at 3′-end S or ASMethyl “Stab 9” Ribo Ribo 5′ and 3′- — Usually S ends “Stab 10” RiboRibo — 1 at 3′-end Usually AS “Stab 11” 2′-fluoro 2′-deoxy — 1 at 3′-endUsually AS Stab 12 2′-fluoro LNA 5′ and 3′- Usually S ends “Stab 13”2′-fluoro LNA 1 at 3′-end Usually AS “Stab 14” 2′-fluoro 2′-deoxy 2 at5′-end Usually AS 1 at 3′-end “Stab 15” 2′-deoxy 2′-deoxy 2 at 5′-endUsually AS 1 at 3′-end “Stab 16 Ribo 2′-O- 5′ and 3′- Usually S Methylends “Stab 17” 2′-O- 2′-O- 5′ and 3′- Usually S Methyl Methyl ends “Stab18” 2′-fluoro 2′-O- 1 at 3′-end Usually AS Methyl CAP = any terminalcap, see for example FIG. 22. All Stab 1-18 chemistries can comprise3′-terminal thymidine (TT) residues All Stab 1-18 chemistries typicallycomprise 21 nucleotides, but can vary as described herein. S = sensestrand AS = antisense strand

TABLE V Peptides for Conjugation SEQ ID Peptide Sequence NO ANTENNAPEDIARQI KIW FQN RRM KWK K amide 507 Kaposi fibroblastAAV ALL PAV LLA LLA P + VQR  508 growth factor KRQ KLMPcaiman crocodylus MGL GLH LLV LAA ALQ GA 509 Ig(5) light chainHIV envelope GAL FLG FLG AAG STM GA + 510 glycoprotein gp41PKS KRK 5 (NLS of the SV40) HIV-1 Tat RKK RRQ RRR 511 InfluenzaGLFEAIAGFIENGWEGMIDGGGYC 512 hemagglutinin  envelop glycoproteinRGD peptide X-RGD-X 513 where X is any amino acid  or peptidetransportan A GWT LNS AGY LLG KIN LKA LAA 514 LAK KIL Somatostatin(S)FC YWK TCT 515 (tyr-3- octreotate) Pre-S-peptide (S)DH QLN PAF 516(S) optional Serine for coupling Italic = optional D isomer forstability

TABLE VI Duplex half-lives in human and mouse serum and liver extractsStability S/AS All RNA 4*/5 4/5* 7/11* 7*/8 7/8* Sirna # 47715/4793330355/30366 30355/30366 30612/31175 30612/30620 30612/30620 Human 0.017408 39 54 130 94 Serum (0.96)^(†) (0.65) (0.76) (0.88) (0.86) t_(1/2)hours Human 2.5 28.6 43.5 0.78/2.9^(‡) 9 816 Liver (0.40) (0.66) (0.45)(0.39) (0.99) t_(1/2) hours Mouse 1.17 16.7 10 2.3 16.6 35.7 Serum (0.9)(0.81) (0.46) (0.69) t_(1/2) hours Mouse 6 1.08 0.80 0.20 0.22 120 Liver(0.89) t_(1/2) hours *The asterisk designates the strand carrying theradiolabel in the duplex. ^(†)For longer half-lives the fractionfull-length at the 18 hours is presented as the parenthetic lower numberin each cell. ^(‡)A biphasic curve was observed, half-lives for bothphases are shown.

TABLE VII Single strand half-lives in human serum Stability Sirna # 4 57 11 8 30355 30366 30612 31175 30620 Human serum 22 16 13 19 28 t_(1/2)hours Human liver 0.92 0.40 0.43 0.27 192 t_(1/2) hours

TABLE VIII Human serum half-lives for Stab 4/5 duplex chemistry with terminus chemistries of FIG. 22 2 7 9 2 8 1 3 6 Cap (R = O) (R = O) (R =O) (R = S) (R = O) (R = O) (R = O) (R = O) Chemistry (B = T) (B = T)(B = T) (B = T) (B = T) (B = T) (B = T) (B = T) Human 1 1.2 2.3 39 96460 770 770 Serum t_(1/2) (0.69)^(‡)  (0.95)  (0.94)  (0.95) hours Thecapping structures were in the following position of the 4:5 chemistryformatted sequence: antisense strand - 5′-uuGuuGuAuuuuGuGGuuG-CAP-3′where CAP is 1, 2, 3, 6, 7, 8, or 9 from FIG. 22. (SEQ ID NO: 670) sensestrand 5′-CAP-cAAccAcAAAAuAcAAcAATT-CAP-3′ where CAP is 1 from FIG. 22.(SEQ ID NO: 671) ^(‡)For half-lives that extend beyond the time coursesampled the fraction full-length is presented in parentheses.

What we claim is:
 1. A compound comprising: (I) a short interferingnucleic acid (siNA) molecule capable of inhibiting expression of a humanhuntingtin gene, wherein the human huntingtin gene comprises anucleotide sequence of GenBank Accession Number NM_002111, said siNAmolecule consisting of a single-stranded polynucleotide comprising anucleotide sequence complementary to a huntingtin RNA, wherein saidsingle-stranded polynucleotide: i) is about 19 to 20 nucleotides inlength; ii) comprises 10 or more phosphorothioate internucleotidelinkages; iii) comprises 10 or more modified pyrimidines chosen from 2′deoxy pyrimidine, 2′-O-methyl pyrimidine, 2′-deoxy-2′-fluoro pyrimidine,locked nucleic acid (LNA) pyrimidine, 2′-methoxyethoxy (MOE) pyrimidine,or a combination thereof; and (II) a galactosamine.
 2. The compound ofclaim 1, wherein the single-stranded polynucleotide comprises 10 or more2′-deoxy pyrimidines.
 3. The compound of claim 1, wherein thesingle-stranded polynucleotide comprises one or more 2′-methoxyethoxy(MOE) pyrimidines.
 4. The compound of claim 1, wherein the galactosaminecomprises N-acetylgalactosamine which is a mono-antennary galactosamine,bi-antennary galactosamine, or a tri-antennary galactosamine.
 5. Thecompound of claim 1, wherein the galactosamine is attached at the 5′-endor the 3′-end of the single-stranded polynucleotide.
 6. The compound ofclaim 1, wherein the single stranded polynucleotide is 20 nucleotideslong.
 7. The compound of claim 1, wherein the 10 or more modifiedpyrimidines comprise 4 or more 2′ deoxy pyrimidines.
 8. A shortinterfering nucleic acid (siNA) molecule capable of inhibitingexpression of a human huntingtin gene, wherein the human huntingtin genecomprises a nucleotide sequence of GenBank Accession Number NM_002111,said siNA molecule consisting of a single-stranded polynucleotidecomprising a nucleotide sequence complementary to a human huntingtinRNA, wherein said single-stranded polynucleotide: i) is about 19 to 20nucleotides in length; ii) comprises 10 or more phosphorothioateinternucleotide linkages; and iii) comprises 10 or more modifiedpyrimidines chosen from 2′ deoxy pyrimidine, 2′-O-methyl pyrimidine,2′-deoxy-2′-fluoro pyrimidine, 2′-methoxyethoxy (MOE) pyrimidine, or acombination thereof.
 9. The siNA molecule of claim 8, wherein thesingle-stranded polynucleotide further comprises a ligand chosen from agalactosamine or a cholesterol.
 10. The siNA molecule of claim 8,wherein the single-stranded polynucleotide further comprises a ligandchosen from a mono-antennary galactosamine, bi-antennary galactosamine,or a tri-antennary galactosamine.
 11. The siNA molecule of claim 8,wherein the single-stranded polynucleotide comprises one or more2′-methoxyethoxy (MOE) pyrimidines.
 12. The siNA molecule of claim 8,wherein the single-stranded polynucleotide comprises 1, 2, 3, 4, 5 ormore locked nucleic acid (LNA) pyrimidines, wherein said LNA pyrimidinesis/are located at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, ofthe single-stranded polynucleotide.
 13. The siNA molecule of claim 8,wherein the 10 or more modified pyrimidines comprise 4 or more 2′ deoxypyrimidines.
 14. The siNA molecule of claim 8, wherein thesingle-stranded polynucleotide comprises 10 or more 2′-deoxynucleotides.
 15. The siNA molecule of claim 8, wherein thesingle-stranded polynucleotide comprises one or more 5-methylcytosines.16. The siNA molecule of claim 8, wherein the single-strandedpolynucleotide comprises 5 or more 5-methylcytosines.
 17. A shortinterfering nucleic acid (siNA) molecule capable of inhibitingexpression of a human huntingtin gene, wherein the human huntingtin genecomprises a nucleotide sequence of GenBank Accession Number NM_002111,said siNA molecule consisting of a single-stranded polynucleotidecomprising a nucleotide sequence complementary to a human huntingtinRNA, wherein said single-stranded polynucleotide: i) is 20 nucleotidesin length; ii) comprises 10 or more phosphorothioate internucleotidelinkages; and iii) comprises 10 or more modified pyrimidines chosen from2′ deoxy pyrimidine, 2′-O-methyl pyrimidine, 2′-deoxy-2′-fluoropyrimidine, 2′-methoxyethoxy (MOE) pyrimidine, or a combination thereof;and iv) comprises one or more 5-methylcytosines.
 18. The siNA moleculeof claim 17, which comprises 5 or more 5-methylcytosine nucleotides. 19.The siNA molecule of claim 17, wherein the single-strandedpolynucleotide comprises one or more 2′-methoxyethoxy (MOE) pyrimidines.20. The siNA molecule of claim 17, wherein the 10 or more modifiedpyrimidines comprise 4 or more 2′ deoxy pyrimidines.
 21. The siNAmolecule of claim 17, wherein the single-stranded polynucleotidecomprises 10 or more modified pyrimidines chosen from 2′ deoxypyrimidine or 2′-methoxyethoxy (MOE) pyrimidine, or a combinationthereof.
 22. The compound of claim 1, wherein at least 60% of nucleotidepositions in the single-stranded polynucleotide are modifiednucleotides.
 23. The compound of claim 1, wherein at least 80% ofnucleotide positions in the single-stranded polynucleotide are modifiednucleotides.
 24. The siNA molecule of claim 8, wherein at least 60% ofnucleotide positions in the single-stranded polynucleotide are modifiednucleotides.
 25. The siNA molecule of claim 8, wherein at least 80% ofnucleotide positions in the single-stranded polynucleotide are modifiednucleotides.
 26. The siNA molecule of claim 17, wherein at least 60% ofnucleotide positions in the single-stranded polynucleotide are modifiednucleotides.
 27. The siNA molecule of claim 17, wherein at least 80% ofnucleotide positions in the single-stranded polynucleotide are modifiednucleotides.